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Enhanced uptake in 2D- and 3D- lung cancer cell models of redox responsive PEGylated nanoparticles with sensitivity to reducing extra-and intracellular environments
Abstract
In the treatment of lung cancer, there is an urgent need of innovative medicines to optimize pharmacological responses of conventional chemotherapeutics while attenuating side effects. Here, we have exploited some relatively unexplored subtle differences in reduction potential, associated with cancer cell microenvironments in addition to the well-known changes in intracellular redox environment. We report the synthesis and application of novel redox-responsive PLGA (poly(lactic-co-glycolic acid)) -PEG(polyethylene glycol) nanoparticles (RR-NPs) programmed to change surface properties when entering tumor microenvironments, thus enhance cell internalization of the particles and their drug cargo. The new co-polymers, in which PEG and PLGA were linked by ‘anchiomeric effector’ dithiylethanoate esters were synthesized by a combination of ring-opening polymerization and Michael addition reactions and employed to prepare NPs. Non redox-responsive nanoparticles (nRR-NPs) based on related PLGA-PEG copolymers were also prepared as comparators. Spherical NPs of around 120 nm diameter with a low polydispersity index and negative zeta potentials as well as good drug loading of docetaxel were obtained. The NPs showed prolonged stability in relevant simulated biological fluids and a high ability to penetrate an artificial mucus layer due to the presence of the external PEG coating. Stability, FRET and drug release studies in conditions simulating intracellular reductive environments demonstrated a fast disassembly of the external shell of the NPs, thus triggering on-demand drug release. FACS measurements and confocal microscopy showed increased and faster uptake of RR-NPs in both 2D- and 3D- cell culture models of lung cancer compared to nRR-NPs. In particular, the ‘designed-in’ reductive instability of RR-NPs in conditioned cell media, the fast PEG release in the extracellular compartment, as well as a diminution of uptake rate in control experiments where extracellular thiols were neutralized, suggested a partial extracellular release of the PEG fringe that promoted rapid internalization of the residual NPs into cells. Taken together, these results provide further evidence of the effectiveness of PEGylated reducible nanocarriers to permeate mucus layer barriers, and establish a new means to enhance cancer cell uptake of drug carriers by extra-and intra-cellular cleavage of protein-and cell-shielding hydrophilic blocks.
... The mucus adhesion study was carried out through mixing 100 μL of liposomes with 2 mL of mucin type II (from porcine stomach) saturated solution at 37 • C [11]. Particle size of the liposomes before and after incubation with mucin solution for 0, 6, 12 and 24 h was measured respectively. ...
... The mucus permeation of different liposomes was measured using a Transwell® based artificial mucus model [5,11]. Briefly, 250 mg of mucin, 250 μL of sterile egg yolk emulsion, 500 mg of DNA, 0.295 mg of DTPA, 250 mg of sodium chloride, 110 mg of potassium chloride and 1 mL of RPMI were added to 50 mL of distilled water, and stirred to get homogeneous artificial mucus [11]. ...
... The mucus permeation of different liposomes was measured using a Transwell® based artificial mucus model [5,11]. Briefly, 250 mg of mucin, 250 μL of sterile egg yolk emulsion, 500 mg of DNA, 0.295 mg of DTPA, 250 mg of sodium chloride, 110 mg of potassium chloride and 1 mL of RPMI were added to 50 mL of distilled water, and stirred to get homogeneous artificial mucus [11]. And 200 μL of the above mucus was added to donor chamber of the Transwell® (pore size: 5 μm; cell diameter: 6.5 mm), with 300 μL of 10 mM, pH 7.40 PBS added to the accepter chamber. ...
Liposome is the promising nanocarrier for pulmonary drug delivery and surface charge is its basic property. However, there is a lack of knowledge about relationship between the liposomal surface charge and its interaction with biological barriers in the lung. Therefore, the purpose of this research is to elucidate the influence of liposome surface charge on its in vivo fate. Firstly, liposomes with positive, negative and neutral surface charge were constructed and characterized, their compatibility towards pulmonary cells was studied. Then their interaction with different biological barriers in lung, including mucus, trachea, bronchoalveolar lavage fluid (BALF) and alveolar macrophage, were investigated. Their retention behavior in lung and systemic exposure were further explored. It was demonstrated that neutrally and negatively charged liposomes were safer than positively charged ones. In the conducting airway, liposome with positive surface charge could better enhance trachea distribution but only within 2 h. In the respiratory region, both neutrally and negatively charged liposomes presented improved mucus permeability, good stability in BALF containing pulmonary surfactant, decreased macrophage uptake, prolonged lung retention and decreased systemic exposure to other organs, with neutrally charged liposome showing superior performance than the negatively charged ones. While the positively charged liposome was not stable in lung microenvironment with aggregation observed, leading to increased alveolar macrophage uptake, thereby lower pulmonary retention and higher risk of systemic exposure. In conclusion, liposomal surface charge is a tunable formulation factor to modulate the interaction with biological barriers in the lung and thus in vivo fate of inhaled liposomes.
... To control the drug release, gatekeeper molecules such as gold NPs and bulky proteins are commonly used to block the pore access, thereby avoiding the premature delivery of the cargo. Depending on the type of gatekeepers, different internal (e.g., pH, redox state, endogenous enzyme) [163][164][165][166] and external (e.g., heat, light, sound, magnetic field) [18,[167][168][169] stimuli are used to induce the opening of the pore outlets of MSNs and the drug release [123]. Upon exposure to the stimulus, these pore keepers are either degraded or bound to silica surfaces via scissile bonds without disintegrating MSNs [25]. ...
... The acidic environment triggers the capped pores resulting in the release of the drug [171,172]. Another internal stimuli, the redox state, is also used for inducing the drug release for the cancer treatment [163]. This stimulation relies on the significant increase in the intracellular glutathione (GSH) level relative to the extracellular medium observed in most cancers. ...
... This stimulation relies on the significant increase in the intracellular glutathione (GSH) level relative to the extracellular medium observed in most cancers. In this approach, the disulfide bonds between the redox-responsive gatekeeper and MSNs are cleaved by GSH, leading to the cap opening and subsequent drug release [163,[173][174][175]. Recently, an interesting study utilized MSNs with gatekeepers responsive to both temperature and pH for a more regulated delivery of chemotherapeutics into lung cancer cells [176]. ...
The efficient and safe delivery of therapeutic drugs, proteins, and nucleic acids are essential for meaningful therapeutic benefits. The field of nanomedicine shows promising implications in the development of therapeutics by delivering diagnostic and therapeutic compounds. Nanomedicine development has led to significant advances in the design and engineering of nanocarrier systems with supra-molecular structures. Smart mesoporous silica nanoparticles (MSNs), with excellent biocompatibility, tunable physicochemical properties, and site-specific functionalization, offer efficient and high loading capacity as well as robust and targeted delivery of a variety of payloads in a controlled fashion. Such unique nanocarriers should have great potential for challenging biomedical applications, such as tissue engineering, bioimaging techniques, stem cell research, and cancer therapies. However, in vivo applications of these nanocarriers should be further validated before clinical translation. To this end, this review begins with a brief introduction of MSNs properties, targeted drug delivery, and controlled release with a particular emphasis on their most recent diagnostic and therapeutic applications.
... These systems have been designed mainly using two different strategies: insertion of a disulfide bond on the polymer backbone chains or the use of reduction-sensitive crosslink molecules which can be incorporated in the core or shell of the F I G U R E 1 Structures of glutathione in its two states encountered in cells: oxidized glutathione (GSSG) reaction occurring through the glutathione peroxidase and reduced glutathione (GSH) through the reaction leaded by the enzyme glutathione reductase. Structures are presented in their fully protonated forms appropriate nanocarriers (Conte et al., 2018;Z. Deng, Yuan, Xu, Liang, & Liu, 2018;Gulfam et al., 2017;Guo et al., 2018;X. ...
... Moreover, disulfide linkers have also been used to promote the connection between two moieties, such as hydrophobic and hydrophilic blocks aiding the formation of amphiphilic polymers. Using this strategy, it is possible to design a micellar-type of carrier that can carry a payload and, when reaching a site of enhanced reduction potential, undergo a change in polymer structure in which the hydrophilic and the hydrophobic blocks separate (Conte et al., 2018;Sauraj et al., 2020;Wang et al., 2017). In turn, this breakdown of an amphiphilic material or particle can be used to release a drug directly or alter the cellular internalization kinetics of the nanoparticle. ...
... For example, Conte et al prepared poly(lactic-co-glycolic acid)-polyethylene glycol based nanoparticles with a disulfide bond attaching the hydrophilic and hydrophobic blocks of the polymer. The authors also prepared nonresponsive nanoparticles as control and the results indicated that the redox responsive nanoparticles were internalized more rapidly, released drugs more effectively in 2D, and penetrated 3D lung cancer spheroids to a greater extent when compared to the control nanoparticles (Conte et al., 2018). ...
Among various types of stimuli‐responsive drug delivery systems, reduction‐responsive polymers have attracted great interest. In general, these systems have high stability in systemic circulation, however, they can respond quickly to differences in the concentrations of reducing species in specific physiological sites associated with a pathology. This is a particularly relevant strategy to target diseases in which hypoxic regions are present, as polymers which are sensitive to in‐situ expressed antioxidant species can, through a local response, release a therapeutic at high concentration in the targeted site, and thus, improve the selectivity and efficacy of the treatment. At the same time, such reduction‐responsive materials can also decrease the toxicity and side effects of certain drugs. To date, polymers containing disulfide linkages are the most investigated of the class of reduction‐responsive nanocarriers, however, other groups such as selenide and diselenide have also been used for the same purpose. In this review article, we discussed the rationale behind the development of reduction‐responsive polymers as drug delivery systems and highlight examples of recent progress. We include the most popular design methods to generate reduction‐responsive polymeric carriers and their applications in cancer therapy, and question what areas may still need to be explored in a field with already a very large number of research articles. Finally, we consider the main challenges associated with the clinical translation of these nanocarriers and the future perspectives in this area.
This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease
Nanotechnology Approaches to Biology > Nanoscale Systems in Biology
Therapeutic Approaches and Drug Discovery > Emerging Technologies
... This system allows for (i) mucus penetration due to the hydrophilic external PEG layer, (ii) reductive cleavage of the disulfide bond by reducing agents at the cancer cell surface, which reduces the outer PEG layer and improves cellular uptake, and (iii) complete removal of the PEG layer by intracellular GSH, leading to NP breakdown and intracellular drug release (Fig. 7). 77 An alternative strategy for improved absorption has been proposed by Le-Vinh et al., using size-shifting nanocarriers. 79 Solid lipid nanoparticles with a phosphate ester and octadecylamine surfactant provided negatively charged NPs that could penetrate the mucus. ...
... These intelligent nanoparticles can be redox-responsive, pHresponsive or ROS-triggered and, subsequently, may change their size, charge or shape accordingly. 77,476,477 Other NP types, such as Janus NPs, may serve as hyper-engineered platforms, leveraging the strengths of different crossing strategies or allow dual-drug treatment with simultaneous delivery in multifunctional NPs. 478,479 While simplicity in formulation has often been regarded as having the greatest translatability from preclinical studies to human clinical trials, these recently suggested engineered formulations may hold the greatest promise for effectively penetrating the barriers of organs. ...
The biomedical use of nanoparticles (NPs) has been the focus of intense research for over a decade. As most NPs are explored as carriers to alter the biodistribution, pharmacokinetics and bioavailability of associated drugs, the delivery of these NPs to the tissues of interest remains an important topic. To date, the majority of NP delivery studies have used tumor models as their tool of interest, and the limitations concerning tumor targeting of systemically administered NPs have been well studied. In recent years, the focus has also shifted to other organs, each presenting their own unique delivery challenges to overcome. In this review, we discuss the recent advances in leveraging NPs to overcome four major biological barriers including the lung mucus, the gastrointestinal mucus, the placental barrier, and the blood-brain barrier. We define the specific properties of these biological barriers, discuss the challenges related to NP transport across them, and provide an overview of recent advances in the field. We discuss the strengths and shortcomings of different strategies to facilitate NP transport across the barriers and highlight some key findings that can stimulate further advances in this field.
... GSH-responsive hydrophilic PEG and hydrophobic poly (lactic acid co glyc acid) (PLGA) copolymer were reported to improve therapy efficacy in lung cance vitro/in vivo. The nanoparticles were spherical with a diameter of around 200 nm negative zeta potential [25]. Redox-responsive PEG with PTX NPs achieved a better tr ment effect than free drugs in a breast cancer xenograft mouse model. ...
... GSH-responsive hydrophilic PEG and hydrophobic poly (lactic acid co glycolic acid) (PLGA) copolymer were reported to improve therapy efficacy in lung cancer in vitro/in vivo. The nanoparticles were spherical with a diameter of around 200 nm and negative zeta potential [25]. Redox-responsive PEG with PTX NPs achieved a better treatment effect than free drugs in a breast cancer xenograft mouse model. ...
Cancer is the leading cause of death in people worldwide. The conventional therapeutic approach is mainly based on chemotherapy, which has a series of side effects. Compared with traditional chemotherapy drugs, nanoparticle-based delivery of anti-cancer drugs possesses a few attractive features. The application of nanotechnology in an interdisciplinary manner in the biomedical field has led to functional nanoparticles achieving much progress in cancer therapy. Nanoparticles have been involved in the diagnosis and targeted and personalized treatment of cancer. For example, different nano-drug strategies, including endogenous and exogenous stimuli-responsive, surface conjugation, and macromolecular encapsulation for nano-drug systems, have successfully prevented tumor procession. The future for functional nanoparticles is bright and promising due to the fast development of nanotechnology. However, there are still some challenges and limitations that need to be considered. Based on the above contents, the present article analyzes the progress in developing functional nanoparticles in cancer therapy. Research gaps and promising strategies for the clinical application are discussed.
... The suspensions of nanoparticles were centrifuged at 5000 rpm for 20 min using centrifuge and the standard curve for AMOT was acquired by UV spectrophotometry. At 275 nm, the released amount (AMOT NPs) was determined spectrophotometrically to determine AMOT NPs entrapment capacity and loading efficiency and the accumulated release percentage (Q%) was calculated as described previously [18,19]. ...
... where, F 0 represents the maximum of the fluorescence emission intensity of the protein in absence of ligand and F the maximum of the fluorescence emission intensity of the protein-ligand complex, Ksv is the Stern-Volmer quenching constant, kq is the bimolecular quenching rate constant τ 0 is the unquenched lifetime, [Q] is the quencher concentration [18]. ...
The drug binding to serum protein (SP) is an area of intense research in evaluating drug candidates. Thus, composite martial based on chitosan (CS), and amoxicillin tri hydrate (AMOT) were designed in order to evaluate the release of [email protected] NPs and it’s binding with BSA under physiological condition. The inclusion of 2 % v/v T80 in the CS NPs enabled in vitro release of AMOT, at 298K and pH 7.4 up to 68.03%. This study also was undertaken to compare and explore the binding of potential chemotherapeutic antibacterial drug Amoxicillin trihydrate (AMOT) and [email protected] nanoparticles ([email protected] NPs) with a model protein bovine serum albumin (BSA) by fluorescence spectroscopy, synchronous fluorescence spectroscopy, Ultraviolet-visible (UV-vis) absorption, and CD spectroscopic techniques. Experimental results indicated that AMOT and ([email protected] NPs) could bind with BSA and quench the fluorescence of BSA via static mechanism. The interactions among BSA with AMOT, AMOT/T80 and [email protected] NPs were evidenced by substantial changes in the BSA secondary structure, as revealed by circular dichroism. The findings of thermodynamic parameters revealed that the binding reaction in both systems was exothermic and spontaneous, enthalpically driven and the hydrogen bonding and Vander Waals forces play a vital role to achieve optimal interaction between AMOT/ [email protected] NPs and BSA.
... Nanocarriers also display improved cellular uptake in comparison to standard chemotherapy drugs. Among the nanocarriers, liposomes, polymeric nanoparticles, and micelles have received the most attention [21]. To date, several nanoparticle-based chemotherapeutics are clinically approved whilst others are in the advanced stages of clinical development. ...
... The physical and chemical properties of NPs greatly influence their efficacy. Nanoscale compounds from synthetic polymers, lipids, proteins, and inorganic particles have been developed [21][22][23][24][25][26][27][28][29]. They promote drug protection, solubility, and stability, enhancing drug delivery. ...
Chemotherapy drugs are cytotoxic to tumor cells, but their lack of specificity leads to a range of side effects. The off-target effects of such drugs can be improved through the use of nanoparticles (NPs). Administered NPs show enhanced accumulation in tumor tissue near the blood vessels, enhancing both anticancer drug permeability and tumor retention. Several nanocarriers are now approved for clinical use in a range of cancer therapies, and many novel formulations are in the later stages of clinical trials. Here, we describe the advances in this area through the review of novel NP drug formulations developed over the last year. We focus specifically on lung, colon, cervical, and breast cancers and discuss the future of NPs as potential treatment options in these areas.
... The capacity of NPs to cross artificial mucus (AM) and a tumor ECM was investigated. The transport experiment was carried out by placing either artificial mucus (AM) (for composition, see SI) or ECM gel (from a murine sarcoma) in the upper chambers of Transwell® -12 well plates (12 mm diameter, polyester membranes with a 3.0 μm pore size), applying NPs, and monitoring the amount of NPs that diffused into the lower chamber [23]. For transport through the AM, the lower chamber was filled with 1 mL of simulated interstitial lung fluid (SILF) (for composition, see Supplementary Information (SI)). ...
... The efficiency of this energy transfer is inversely proportional to the sixth power of the distance between the donor and acceptor, making FRET extremely sensitive to small changes in distance. Measurement of FRET efficiency is a useful tool for determining if two fluorophores are released from an NP system and to monitor its disassembly in different biological conditions, in vitro and in vivo [23,30]. As donor and acceptor chromophores, we selected the hydrophobic dyes DiO (λex/em is 488/505 nm) and DiL (λex/em at 543/575 nm), respectively, since the DiO emission spectrum overlaps well with the DiL absorption spectrum. ...
Nanoparticles (NPs) based on amphiphilic block copolymers of polyethylene glycol (PEG) and biodegradable polyesters are of particular current interest in drug nanodelivery due to their easily manipulated properties. The interaction of these NPs with biological environments is highly influenced by shell features, which drive biological identity after administration. To widen the strategies available for tuning particle surface chemistry, here we developed a panel of amine-bearing PEGylated NPs with a poly(ε-caprolactone) (PCL) core for the delivery of lipophilic drugs, and investigated the impact of NP modifications on their interaction with abundant circulating proteins (human serum albumin-HSA-and mucin), as well as their transport through biological barriers (artificial mucus-AM, extracellular matrix-ECM). We prepared NPs based on a diamino-terminated PCL (amine-NPs) and its mixture with PEG-PCL copolymers (amine/PEG-NPs) at different PEG molecular weights by nanoprecipitation, as well as corresponding NPs of PEG-PCL (PEG-NPs). The presence of an amine-bearing polymer resulted in NPs with a net positive charge and a zeta potential dependent on the length of PEG in the copolymer. Amine/PEG-NPs had a larger fixed aqueous layer thickness as compared to PEG-NPs, suggesting that PEG conformation is affected by the presence of positive charges. In general, amine-bearing NPs promptly interacted with the dysopsonic protein HSA, due to electrostatic interactions, and lose stability, thereby undergoing time-related aggregation. On the other hand, amine/PEG-NPs interaction with mucin induced switching to a negative surface charge but did not alter the quality of the dispersion. The transport kinetics of NPs through a layer of artificial mucus and tumor extracellular matrix was studied by means of fluorescent NPs based upon FRET. Amine/PEG-NPs did not cross the ECM, but they were promptly transported through the AM, with swifter transport noted at increasing MWs of PEG in the copolymer. Finally, we demonstrated that all the different NP types developed in this study are internalized by human monocytes and, despite the positive charge, they did not induce a measurable inflammatory effect. In conclusion, we showed that the concurrent presence of both PEG and amine groups on NP surface is a promising strategy for directing their interaction with body compartments. While PEG-NPs are confirmed for their capacity to cross ECM-like compartments, amine/PEG-NPs are revealed as a powerful platform to widen the arsenal of nanotools available for overcoming mucus-covered epithelia.
... Although there have been improvements in diagnosis and treatment, the prognosis of lung cancer patients remains poor. Physiological and pathological barriers of the lung including the TME, which varies among individuals and types of cancer, are the biggest hurdles in treating lung cancer [58]. Recently, there has been significant progress in targeting TAMs to develop novel anti-tumoral therapeutic strategies, with encouraging results. ...
... TAMs are mostly found in the lung TME [58]. Multiple studies have indicates that TAMs are predominantly of the M2 phenotype in patients with NSCLC associated with lower survival rate [87]. ...
Tumor-associated macrophages (TAMs) represent as much as 50% of the solid mass in many different types of human solid tumors including lung, breast, ovarian, and pancreatic adenocarcinomas. The tumor microenvironment plays an important role in the polarization of macrophages into the M1 phenotype, which is tumor-suppressive, or M2 phenotype, which is tumor promoting. Preclinical and clinical evidence suggests that TAMs are predominantly of the M2 phenotype that support immune suppression, tumor growth, angiogenesis, metastasis and therapeutic resistance. Hence, significant attention has been focused on the development of strategies for the modification of TAMs to halt lung cancer progression. The promotion of repolarization from the M2 to the M1 subtype, or the prevention of M2 polarization of TAMs in the stromal environment are potential approaches to reduce progression and metastasis of lung cancer. The focus of this article is an introduction to the development and evaluation of therapeutic agents that may halt lung cancer progression via the manipulation of macrophage polarization. The article will address recent advances in the therapeutic efficacy of nanomedicine exploiting surface functionalization of nanoparticles and will also consider future perspectives.
... Tumor cells have higher GSH, breaking these bonds, disassembling nanocarriers, and releasing payloads. Cysteine, cystamine, and dimethacrylate are frequent precursors for polymeric backbone disulfide linkages (Guo et al. 2018;Conte et al. 2018;Monteiro et al. 2021;Peng et al. 2019). ...
Stimuli-responsive polymers (smart polymers) are macromolecules that change their physicochemical properties in response to specific triggers from the external environment (e.g., heat, light, electrical or magnetic fields), and endogenous stimuli (e.g., redox reactions). In recent years, drug delivery and diagnosis strategies have had enormous applications of nanocarriers of intelligent polymers. A significant number of natural polymers, synthetic copolymers, and block copolymers have been employed in fabricating biodegradable nanoparticles for various biomedical applications. Block copolymers are formed by joining two or more monomer blocks in a chain. The recent developments in the synthesis of block copolymers have made it possible to create versatile intelligent polymers that can be tailored to the specific needs of various applications. The physicochemical properties of these polymers are quite different from the polymers of natural origin. As the chains of these block copolymers are composed of a variety of monomers, each of which possesses its unique properties, it is possible to use them in targeted and controlled drug delivery as well as in other biomedical applications by employing a combination of endogenous and exogenous stimulation. This chapter is focused on the synthesis, chemistry, physicochemical properties, characterization, and applications of multi-block copolymers that are responsive to pH, temperature, and redox potential. Special care is taken to elaborate the application of such polymers in developing multifunctional nanoparticles for cancer therapy.
... Furthermore, due to rapid enzymatic degradation in human plasma, the concentration of glutathione (GSH) in the cytosol and subcellular compartments (such as lysosomes and endosomes) is about 2-10 mM, which is approximately 100-1000 times higher than that in the cellular exterior, which is about 2-10 μM [26][27][28][29][30]. A disulfide bond (-S-S-) is a type of reversible covalent connection that is created when two thiol groups are oxidized. ...
Present cancer treatment using chemotherapy is limited owing to serious adverse effects on normal cells. To manage this problem, targeted drug delivery using smart polymeric nanoparticles and/or mesoporous silica nanoparticles can play a key role. In this study, a combination of surfactant-directed sol-gel and seeded precipitation polymerization techniques were designed to synthesize hyaluronic acid-decorated pH and redox dual-stimuli responsive hollow mesoporous organosilica/poly(methacrylic acid) nanospheres for active-targeted delivery of curcumin to breast cancer cells. The obtained nanospheres possess diameter less than 200 nm, high negative zeta potential (−30 mV), and narrow size distribution. The prepared nanospheres exhibited a high entrapment efficiency up to 70 % and drug loading capacity more than 10 % for curcumin. In vitro drug release studies showed that, the cumulative drug release was remarkably restricted under normal physiological media (neutral pH and in the absence of glutathione), while it was accelerated at the simulated tumor tissue conditions (acidic pH and in the presence of glutathione), indicating redox and pH-responsivity of the prepared nanocarrier. In vitro cytotoxicity and apoptosis assays demonstrated that the empty nanospheres have excellent biocompatibility, and curcumin-loaded targeted nanospheres are more cytotoxic against MCF-7 human breast cancer cells compared to the free drug and non-targeted nanospheres.
... The physical and chemical characteristics of NPs have a significant impact on their efficacy. Nanoscale molecules have been synthesized using a variety of materials, including synthetic lipids, proteins, polymers, and inorganic particles [34][35][36][37][38][39][40][41][42]. They provide drug protection, solubility, and stability, which improves pharmaceutical distribution. ...
Background The second leading cause of mortality in the world, behind cardiovascular disorders, is cancer. The currently employed treatment options including radiotherapy, chemotherapy are reported with many adverse reactions. These limitations in combination with high cost of therapy have restricted the management of malignancy. In this review, several nanocarriers-based approaches were described as effective treatment option of malignancy.
The main body of the abstract The development of innovative and effective targeted therapies for malignancy relies on alterations in the molecular biology of cancerous cells. Given the nonselective destruction of healthy cells, the harmful effects of existing chemotherapy drugs, and the development of multidrug resistance, has thrived the development of novel carriers for improved targeting efficacy of anticancer drugs. The present study offers a comprehensive account of diverse cytotoxic drug carriers, such as carbon nanotubes, liposomes, polymeric micelles,
dendrimers, polymeric nanoparticles, and polymeric conjugates, in the context of passive and active targeted cancer therapy. The carriers are known to enhance the permeability and retention or functionalize the surface, thereby improving the efficacy of drug delivery.
Short conclusion The present literature delineates the progressions made in the nanoengineered approach for administering therapeutic agents to the tumour micro-environment.
... As such, we performed cell viability studies in 3D cell models of the same A549 cell line since these can overcome some shortcomings of the 2D cancer cell cultures and better mimic the in vivo acidic and reductive tumour microenvironment. [48][49][50][51] The results in Figure 5C show that there was no significant difference in the cell viability following treatment with cisplatin, the Pt(IV) prodrug 1 and MnO2-Pt(IV) nanoparticles (100 μM of Pt) in 3D cell systems, demonstrating that these more complex 3D cell systems better recapitulate the reductive in vivo tumour microenvironment. The ability of the released Pt(II) drug to interact with DNA was also explored. ...
Manganese dioxide (MnO2)-based nanostructures have emerged as promising tumour microenvironment (TME) responsive platforms. Herein, we used a one-pot reaction to prepare MnO2 nanostructures with Pt(IV) prodrugs as redox- (and thus TME-) responsive theranostics for cancer therapy, in which the Pt(IV) complexes act as prodrugs of cisplatin (Pt(II)), a clinical chemotherapeutic drug. The cytotoxicity of these MnO2-Pt(IV) probes was evaluated in two and three dimensional (2D and 3D) A549 cell models and found to be as effective as active drug cisplatin in 3D models. Moreover, MnO2-Pt(IV) nanoparticles exhibited strong off/ON magnetic resonance (MR) contrast in response to reducing agents, with the longitudinal relaxivity (r1) increasing 136-fold upon treatment with ascorbic acid. This off/ON MR switch was also observed in (2D and 3D) cells in vitro. In vivo MRI experiments revealed that the nanostructures induce a strong and long-lasting T1 signal enhancement upon intratumoral injection in A549 tumour-bearing mice. These results show the potential of MnO2-Pt(IV) NPs as redox responsive MR theranostics for cancer therapy.
... 6-10 Nanoparticles as therapeutic carriers for lung cancer are appealing because their unique size and structure designs allow them to maintain stability, cross biological barriers, 11 bind to multiple targeting ligands, 12 reduce non targeting cytotoxic effects of drugs, 13 and improve circulation times with improved loading capacity 14 and releasing efficiency. [15][16][17][18] Although, targeted delivery of modified nanocarriers to lung cells has been developed with the purpose of improving therapeutic outcomes, [19][20][21][22][23] few have reached clinical relevance 24 due in part to the unique delivery challenge that is imparted by the physical environment of the lung and absence of efficient alternative options. ...
The mechanical properties and forces in the extracellular environment surrounding alveolar epithelial cells have the potential to modulate their behavior. Particularly, breathing applies 3-dimensional cyclic stretches to the cells, while the stiffness of the interstitium changes in disease states, such as fibrosis and cancer. A platform was developed that effectively imitates the active forces in the alveolus, while allowing one to control the interstitium matrix stiffnesses to mimic fibrotic lung tumor microenvironments. Alveolar epithelial cancer cells were cultured on these platforms and changes in the glycocalyx expression were evaluated. A complex combination of stiffness and dynamic forces altered heparan sulfate and chondroitin sulfate proteoglycan expressions. Consequently, we designed liposomal nanoparticles (LNPs) modified with peptides that can target heparan sulphate and chondroitin sulfates of cell surface glycocalyx. Cellular uptake of these modified nanoparticles increased in stiffer conditions depending on the stretch state. Namely, chondroitin sulfate A targeting improved uptake efficiency in cells experiencing dynamic stretches, while cells seeded on static stiff interstitium preferentially took up heparan sulfate targeting LNPs. These results demonstrate the critical role that mechanical stiffness and stretching play in the alveolus and the importance of including these properties in nanotherapeutic design for cancer treatment.
... The stability of liposomes was evaluated in a saturated mucin dispersion (0.8 % w/v) prepared as previously described (Conte et al., 2018). Variations of mean diameter and polydispersity index were assessed after their preparation (0 h) and at 2, 4 and 24 h. ...
The Echium amoenum Fisch. and C.A. Mey. (E. amoenum) is an herb native from Iranian shrub, and its blue-violet flowers are traditionally used as medical plants. In the present study, an antioxidant phytocomplex was extracted from the flowers of E. amoenum by ultrasounds-assisted hydroalcoholic maceration. The main components, contained in the extract, have been detected using HPLC-DAD, and rosmarinic acid was found to be the most abundant. The antioxidant power of the extract along with the phenolic content were measured using colorimetric assays. The extract was loaded in liposomes, which were enriched adding different bioadhesive polymers (i.e., mucin, xanthan gum and carboxymethyl cellulose sodium salt) individually or in combination. The main physico-chemical properties (i.e. size, size distribution, surface charge) of the prepared vesicles were measured as well as their stability on storage. The viscosity of dispersion and the ability of vesicles to interact with mucus were evaluated measuring their stability in a mucin dispersion and mobility in a mucin film. The biocompatibility and the ability of the formulations to protect keratinocytes from damages caused by hydrogen peroxide and to promote the cell migration were measured in vitro.
... In this research, non-redox-responsive NPs (nRR-NPs) and redox-responsive NPs (RR-NPs) were obtained by PLGA-PEG and PLGA-S-S-PEG, respectively. Comparative tests between nRRNPs and RR-NPs displayed that RR-NPs had increased uptake and faster degradation in lung cancer models as a result of the decomposition of the disulfide bonds by GSH [105]. ...
Cancer is one of the leading causes of death worldwide, and battling cancer has always been a challenging subject in medical sciences. All over the world, scientists from different fields of study try to gain a deeper knowledge about the biology and roots of cancer and, consequently, provide better strategies to fight against it. During the past few decades, nanoparticles (NPs) have attracted much attention for the delivery of therapeutic and diagnostic agents with high efficiency and reduced side effects in cancer treatment. Targeted and stimuli-sensitive nanoparticles have been widely studied for cancer therapy in recent years, and many more studies are ongoing. This review aims to provide a broad view of different nanoparticle systems with characteristics that allow them to target diverse properties of the tumor microenvironment (TME) from nanoparticles that can be activated and release their cargo due to the specific characteristics of the TME (such as low pH, redox, and hypoxia) to nanoparticles that can target different cellular and molecular targets of the present cell and molecules in the TME.
... Several chemotherapeutics based on nanoparticles have received clinical approval to date, while others are in the advanced phases of clinical trials. However, nanocarriers have certain disadvantages, including poor biodegradability, bioavailability, stability, and tissue distribution, as well as high toxicity, which raise safety concerns, especially for long-term cancer therapy [18][19][20]. ...
Curcumin (Cur) is a traditional herb with known anticancer properties against various malignancies such as breast cancer. In this study, a metal-organic framework (MOF) with excellent water stability based on UIO-66 was prepared to deliver Cur into MDA-MB-231 and SKBR3 human breast cancer cell lines. The size of the fabricated UIO-66 ranged from 100 to 150 nm with a spherical shape and triangular base pyramid morphology. Favorable adsorption of Cur on the UIO-66 was approved by Fourier transform infrared spectroscopy (FTIR). The release profile exhibited a slow pH-dependent release of Cur from UIO-66 with significant enhancement in the acidic microenvironment (50, 55, and 70% Cur release from UIO-66-Cur in pH values of 7.4, 6.5, and 5.4 in 72 h test), showing UIO-66-Cur versatile releasing capacity in the acidic milieu of breast cancer. The characterization and structural elucidation of nanoparticles were evaluated by N2 adsorption–desorption, thermogravimetric analysis (TGA), differential thermogravimetric (DTG), Brunauer Emmett-Teller (BET), Barrett-Joyner Halenda (BJH), and adsorption kinetics that showed promising results. Particularly, the UIO-66-Cur exhibited the highest adsorption capacity for Cur. The kinetics of drug adsorption were investigated by three well-known kinetic models, which the output indicated that the adsorption of drug into the synthesized MOFs followed the pseudo-second-order model. UIO-66's TGA and DTG curves demonstrated a three-step weight reduction. The N2 adsorption/desorption isotherms and pore size distribution characteristics of UIO-66. The BET surface areas, and total pore volumes of UIO-66 were computed to be 910.58 m²/g, and 0.49 cm³/g. The size, and entrapment efficacy (EE) of nanoparticles were more stable at 4 °C compared to 25 °C. Furthermore, the cytotoxic effects of UIO-66-Cur against MDA-MB-231 and SKBR3 cell lines were demonstrated by using MTT assay, flow cytometry, gene expression profile, migration assay, and DAPI staining. The MTT assay revealed the high biocompatibility of UIO-66 with MCF10A healthy breast cell line (95% viability at concentration of 200 μg/ml), whereas UIO-66-Cur showed cytotoxicity toward MCF10A at high concentrations (78% viability at concentration of 200 μg/ml). Furthermore, loading of Cur into UIO-66 notably increased its anticancer activity as the IC50 of UIO-66-Cur was significantly lower than Cur (72.2 vs. 159.3 against SKBR3 and 109.3 vs. 269.1 against MD-MBA-231 in 72 h test). It was shown that loading of Cur into UIO-66 profoundly promotes its anticancer activity as UIO-66-Cur significantly induced caspase 3 and caspase 9 and inhibited MMP-2, MMP-9 and cyclin E/D expression in cancer cell lines.
... Such nanoparticles not only showed a faster uptake in lung cancer in vitro models, they also allowed the sustained release of loaded therapeutics when compared to the control group (i.e., nanoparticles made of PLGA-PEG di-block copolymer without disulfide bonds). When exposed to glutathione, a fast and triggered release of loaded therapeutics was observed for redox-responsive nanoparticles, indicating PEG cleavage, PLGA degradation and disassembly of nanoparticles due to the presence of glutathione [323]. Other co-polymers designed with a similar strategy were described in work by Shen et al., in which hyaluronic acid-modified disulfide was crosslinked with PLGA-polyethyleneimine. ...
Although nanomedicine has been highly investigated for cancer treatment over the past decades, only a few nanomedicines are currently approved and in the market; making this field poorly represented in clinical applications. Key research gaps that require optimization to successfully translate the use of nanomedicines have been identified, but not addressed; among these, the lack of control of the release pattern of therapeutics is the most important. To solve these issues with currently used nanomedicines (e.g., burst release, systemic release), different strategies for the design and manufacturing of nanomedicines allowing for better control over the therapeutic release, are currently being investigated. The inclusion of stimuli-responsive properties and prolonged drug release have been identified as effective approaches to include in nanomedicine, and are discussed in this paper. Recently, smart sustained release nanoparticles have been successfully designed to safely and efficiently deliver therapeutics with different kinetic profiles, making them promising for many drug delivery applications and in specific for cancer treatment. In this review, the state-of-the-art of smart sustained release nanoparticles is discussed, focusing on the design strategies and performances of polymeric nanotechnologies. A complete list of nanomedicines currently tested in clinical trials and approved nanomedicines for cancer treatment is presented, critically discussing advantages and limitations with respect to the newly developed nanotechnologies and manufacturing methods. By the presented discussion and the highlight of nanomedicine design criteria and current limitations, this review paper could be of high interest to identify key features for the design of release-controlled nanomedicine for cancer treatment.
... In vitro drug release from GUL-SS DTX/EZL-NPs was evaluated in phosphate buffer solution (PBS) with or without GSH (10 mM) by a dialysis method (Conte et al., 2018). GUL-SS DTX/EZL-NPs was dispersed in PBS and placed in a dialysis bag (molecular weight cutoff of 3500 Da). ...
Prostate cancer (PCa) is the most common malignant tumor in men. Chemotherapy with docetaxel (DTX) and novel hormonal agents such as enzalutamide (EZL) and abiraterone are the preferred first-line therapeutic regimens. Prostate-specific membrane antigen (PSMA) is overexpressed on the surface of PCa cells. This study aimed to prepare a PSMA targeted (Glutamate-Urea-Lysine, GUL ligand modified), glutathione (GSH)-sensitive (Cystamine, SS), DTX and EZL co-loaded nanoparticles (GUL-SS DTX/EZL-NPs) to treat PCa. Polyethylene glycol (PEG) was conjugated with oleic acid (OA) using a GSH-sensitive ligand: cystamine (PEG-SS-OA). GUL was covalently coupled to PEG-SS-OA to achieve GUL-PEG-SS-OA. GUL-PEG-SS-OA was used to prepare GUL-SS DTX/EZL-NPs. To evaluate the in vitro and in vivo efficiency of the system, human prostate cancer cell lines and PCa cells bearing mice were applied. Single drug-loaded nanoparticle and free drugs systems were utilized for the comparison of the anticancer ability. GUL-SS DTX/EZL-NPs showed a size of 143.7 ± 4.1 nm, with a PDI of 0.162 ± 0.037 and a zeta potential of +29.1 ± 2.4 mV. GUL-SS DTX/EZL-NPs showed high cancer cell uptake of about 70%, as well as higher cell growth inhibition efficiency (a maximum 79% of cells were inhibited after treatment) than single drug-loaded NPs and free drugs. GUL-SS DTX/EZL-NPs showed the most prominent tumor inhibition ability and less systemic toxicity. The novel GUL-SS DTX/EZL-NPs could be used as a promising system for PCa therapy.
... The acidic environment of the cancer cells stimulates the covered pores and leads to drug release [27]. The redox state is an internal stimulus that is also used for the stimulation of drug release for cancer treatment [120]. This stimulation is based on the remarkable increase in the intracellular GSH level, compared to the extracellular environment, discerned in most cancers. ...
This review focuses on the biomedical application of mesoporous silica nanoparticles (MSNs), mainly focusing on the therapeutic application of MSNs for cancer treatment and specifically on overcoming the challenges of currently available anthelmintics (e.g., low water solubility) as repurposed drugs for cancer treatment. MSNs, due to their promising features, such as tunable pore size and volume, ability to control the drug release, and ability to convert the crystalline state of drugs to an amorphous state, are appropriate carriers for drug delivery with the improved solubility of hydrophobic drugs. The biomedical applications of MSNs can be further improved by the development of MSN-based multimodal anticancer therapeutics (e.g., photosensitizer-, photothermal-, and chemotherapeutics-modified MSNs) and chemical modifications, such as poly ethyleneglycol (PEG)ylation. In this review, various applications of MSNs (photodynamic and sonodynamic therapies, chemotherapy, radiation therapy, gene therapy, immunotherapy) and, in particular, as the carrier of anthelmintics for cancer therapy have been discussed. Additionally, the issues related to the safety of these nanoparticles have been deeply discussed. According to the findings of this literature review, the applications of MSN nanosystems for cancer therapy are a promising approach to improving the efficacy of the diagnostic and chemotherapeutic agents. Moreover, the MSN systems seem to be an efficient strategy to further help to decrease treatment costs by reducing the drug dose.
... Nanocarriers also have superior cellular uptake as compared to standard chemotherapeutic molecules. Among the nanocarriers, polymeric NPs, micelles, and liposomes have received significant attention (7). ...
Nowadays, effective cancer therapy is a global concern, and recent advances in nanomedicine are crucial. Cancer is one of the major fatal diseases and a leading cause of death globally. Nanotechnology provides rapidly evolving delivery systems in science for treating diseases in a site-specific manner using natural bioactive compounds, which are gaining widespread attention. Nanotechnology combined with bioactives is a very appealing and relatively new area in cancer treatment. Natural bioactive compounds have the potential to be employed as a chemotherapeutic agent in the treatment of cancer, in addition to their nutritional benefits. Alginate, pullulan, cellulose, polylactic acid, chitosan, and other biopolymers have been effectively used in the delivery of therapeutics to a specific site. Because of their biodegradability, biopolymeric nanoparticles (BNPs) have received a lot of attention in the development of new anticancer drug delivery systems. Biopolymer-based nanoparticle systems can be made in a variety of ways. These systems have developed as a cost-effective and environmentally friendly solution to boost treatment efficacy. Effective drug delivery systems with improved availability, increased selectivity, and lower toxicity are needed. Recent research findings and current knowledge on the use of BNPs in the administration of bioactive chemicals in cancer therapy are summarized in this review.
... To create these systems, a polymer with a disulfide bond (S-S) or molecules with a reduction-sensitive sulfhydryl (thiol) cross-link is embedded in the core or shell of suitable nanocarriers. Due to the elevated amounts of reduced GSH in tumour cells, disulfide linkages are broken down into sulfhydryl groups, resulting in carrier disassembly and payload release (Coen et al., 2016;Conte et al., 2018;Luo et al., 2022;Monteiro et al., 2021Monteiro et al., , 2020. GSH molecule's thiol group (SH) is essential for conjugation and reduction reactions. ...
Cancer is one of the most challenging, life-threatening illnesses to cure, with over 10 million new cases diagnosed each year globally. Improved diagnostic cum treatment with common side-effects are warranting for successful therapy. Nanomaterials are recognized to improve early diagnosis, imaging, and treatment. Recently, multifunctional nanocomposites attracted considerable interest due to their low-cost production, and ideal thermal and chemical stability, and will be beneficial in future diagnostics and customized treatment capacity. Stimuli-Responsive Hybrid Metal Nanocomposites (SRHMNs) based nanocomposite materials pose the on/off delivery of bioactive compounds such as medications, genes, RNA, and DNA to specific tissue or organs and reduce toxicity. They simultaneously serve as sophisticated imaging and diagnostic tools when certain stimuli (e.g., temperature, pH, redox, ultrasound, or enzymes) activate the nanocomposite, resulting in the imaging-guided transport of the payload at defined sites. This review in detail addresses the recent advancements in the design and mechanism of internal breakdown processes of the functional moiety from stimuli-responsive systems in response to a range of stimuli coupled with metal nanoparticles. Also, it provides a thorough understanding of SRHMNs, enabling non-invasive interventional therapy by resolving several difficulties in cancer theranostics.
... In a recent in vitro study, redox-sensitive poly(lacticco-glycolic acid) (PLGA)-PEG NPs were used to deliver DTX into lung cancer cells via the pulmonary route. The 2D and 3D cell culture models showed that redox-responsive NPs were more effective than non-redox-responsive NPs ( Figure 7A) [95]. Lyer et al. developed cisplatin-loaded GSH-sensitive NPs (CGPU) to effectively target lung cancer. ...
Lung cancer is a significant health problem worldwide. Unfortunately, current therapeutic strategies lack a sufficient level of specificity and can harm adjacent healthy cells. Consequently, to address the clinical need, novel approaches to improve treatment efficiency with minimal side effects are required. Nanotechnology can substantially contribute to the generation of differentiated products and improve patient outcomes. Evidence from previous research suggests that nanotechnology-based drug delivery systems could provide a promising platform for the targeted delivery of traditional chemotherapeutic drugs and novel small molecule therapeutic agents to treat lung cancer cells more effectively. This has also been found to improve the therapeutic index and reduce the required drug dose. Nanodrug delivery systems also provide precise control over drug release, resulting in reduced toxic side effects, controlled biodistribution, and accelerated effects or responses. This review highlights the most advanced and novel nanotechnology-based strategies, including targeted nanodrug delivery systems, stimuli-responsive nanoparticles, and bio-nanocarriers, which have recently been employed in preclinical and clinical investigations to overcome the current challenges in lung cancer treatments.
... GSH is a common internal environmental stimulus in cells that rapidly destroys the stability of intracellular nanocarriers to achieve effective drug release (Cheng et al., 2011) (Figure 4A). Several researchers have focused on developing functional carriers with reduction responsiveness based on two strategies: insertion of disulfide bonds in the polymer backbone or using reductionsensitive cross-linking molecules (Gulfam et al., 2017;Conte et al., 2018;Deng et al., 2018;Monteiro et al., 2020). Glutathione (GSH) levels inside and outside cancer cells are markedly different (Lai et al., 2021;Liu et al., 2021). ...
Bladder cancer is one of most common malignant urinary tract tumor types with high incidence worldwide. In general, transurethral resection of non-muscle-invasive bladder cancer followed by intravesical instillation of chemotherapy is the standard treatment approach to minimize recurrence and delay progression of bladder cancer. However, conventional intravesical chemotherapy lacks selectivity for tumor tissues and the concentration of drug is reduced with the excretion of urine, leading to frequent administration and heavy local irritation symptoms. While nanomedicines can overcome all the above shortcomings and adhere to the surface of bladder tumors for a long time, and continuously and efficiently release drugs to bladder cancers. The rapid advances in targeted therapy have led to significant improvements in drug efficacy and precision of targeted drug delivery to eradicate tumor cells, with reduced side-effects. This review summarizes the different available nano-systems of targeted drug delivery to bladder cancer tissues. The challenges and prospects of targeted therapy for bladder cancer are additionally discussed.
... This new finding showed rapid cellular uptake, penetration, and subsequent docetaxel release than conventional PEGylated liposomal system. Another delivery system based on redox-responsive PEGylated strategy is reported by Conte et al. [68]. PLGAPEG amphiphilic blocks linked by dithiylethanoate esters have been programmed to change surface properties when invading the tumor microenvironments, with the loss of the PEG shell and the PLGA core degradation. ...
In recent years the worldwide research community has highlighted innumerable benefits of nanomaterials in cancer detection and therapy. Nevertheless, the development of cancer nanomedicines and other bionanotechnology requires a huge amount of considerations about the interactions of nanomaterials and biological systems, since long-term effects are not yet fully known. Open issues remain the determination of the nanoparticles distributions patterns and the internalization rate into the tumor while avoiding their accumulation in internal organs or other healthy tissues. The purpose of this work is to provide a standard overview of the most recent advances in nanomaterials to fight cancer and to collect trends and future directions to follow according to some critical aspects still present in this field. Complementary to the very recent review of Wolfram and Ferrari which discusses and classifies successful clinically-approved cancer nanodrugs as well as promising candidates in the pipeline, this work embraces part of their proposed classification system based on the exploitation of multifunctionality and extends the review to peer-reviewed journal articles published in the last 3 years identified through international databases.
... For example, Conte et al. developed novel redox-responsive PLGA (poly(lactic-co-glycolic acid))-PEG (polyethylene glycol) nanoparticles (RR-NPs) and compared them to corresponding non-redox-responsive nanoparticles (nRR-NPs) in terms of penetration ability into A549 spheroids. Significant penetration of RR-NPs was achieved through all regions of the spheroids (the outer proliferating zone, the middle viable layer of quiescent cells and the central necrotic core) when compared to nRR-NPs [64]. Disease-specific cell marker expression is usually recapitulated more accurately by 3D cell spheroids than by 2D monolayer cultures. ...
Complex three-dimensional (3D) cell cultures are being increasingly implemented in biomedical research as they provide important insights into complex cancer biology, and cell-cell and cell-matrix interactions in the tumor microenvironment. However, most methods used today for 3D cell culture are limited by high cost, the need for specialized skills, low throughput and the use of unnatural culture environments. We report the development of a unique biomimetic hydrogel microwell array platform for the generation and stress-free isolation of cancer spheroids. The poly N-isopropylacrylamide-based hydrogel microwell array (PHMA) has thermoresponsive properties allowing for the attachment and growth of cell aggregates/ spheroids at 37 °C, and their easy isolation at room temperature (RT). The reversible phase transition of the microwell arrays at 35 °C was confirmed visually and by differential scanning calorimetry. Swelling/ shrinking studies and EVOS imaging established that the microwell arrays are hydrophilic and swollen at temperatures <35 °C, while they shrink and are hydrophobic at temperatures >35 °C. Spheroid development within the PHMA was optimized for seeding density, incubation time and cell viability. Spheroids of A549, HeLa and MG-63 cancer cell lines, and human lung fibroblast (HLF) cell line generated within the PHMAs had relatively spherical morphology with hypoxic cores. Finally, using MG-63 cell spheroids as representative models, a proof-of-concept drug response study using doxorubicin hydrochloride was conducted. Overall, we demonstrate that the PHMAs are an innovative alternative to currently used 3D cell culture techniques, for the high-throughput generation of cell spheroids for disease modeling and drug screening applications.
... However, when exposing the already formed spheroids for 24 h, NPs were seen only to a depth of 20 µm [26]. Cell types, cell densities, physicochemical characteristics of the NPs (including size distribution) and ion release may influence the penetration inside the spheroid [73][74][75][76][77]. ...
(1) In compliance with the 3Rs policy to reduce, refine and replace animal experiments, the development of advanced in vitro models is needed for nanotoxicity assessment. Cells cultivated in 3D resemble organ structures better than 2D cultures. This study aims to compare cytotoxic and genotoxic responses induced by titanium dioxide (TiO2), silver (Ag) and zinc oxide (ZnO) nanoparticles (NPs) in 2D monolayer and 3D spheroid cultures of HepG2 human liver cells. (2) NPs were characterized by electron microscopy, dynamic light scattering, laser Doppler anemometry, UV-vis spectroscopy and mass spectrometry. Cytotoxicity was investigated by the alamarBlue assay and confocal microscopy in HepG2 monolayer and spheroid cultures after 24 h of NP exposure. DNA damage (strand breaks and oxidized base lesions) was measured by the comet assay. (3) Ag-NPs were aggregated at 24 h, and a substantial part of the ZnO-NPs was dissolved in culture medium. Ag-NPs induced stronger cytotoxicity in 2D cultures (EC50 3.8 µg/cm2) than in 3D cultures (EC50 > 30 µg/cm2), and ZnO-NPs induced cytotoxicity to a similar extent in both models (EC50 10.1–16.2 µg/cm2). Ag- and ZnO-NPs showed a concentration-dependent genotoxic effect, but the effect was not statistically significant. TiO2-NPs showed no toxicity (EC50 > 75 µg/cm2). (4) This study shows that the HepG2 spheroid model is a promising advanced in vitro model for toxicity assessment of NPs.
... However, the therapeutic potential of gene therapy is yet to be realized completely due to the challenges associated with stability, target specificity and transfection [1]. The use of viral or non-viral vectors to deliver the therapeutic oligonucleotide to the target cell has been widely explored to overcome the inherent problems associated with the administration of the naked oligonucleotide [2]. The majority of gene delivery studies have employed viral vectors due to their superior transfection capabilities. ...
The present work explores the ability of poly(1-vinylimidazole) (PVI) to complex small interfering RNA (siRNA) silencing vascular endothelial growth factor (VEGF) and the in vitro efficiency of the formed complexes in A549 lung cancer cells. The polyplex formed was found to exhibit 66% complexation efficiency. The complexation was confirmed by gel retardation assays, FTIR and thermal analysis. The blank PVI polymer was not toxic to cells. The polyplex was found to exhibit excellent internalization and escaped the endosome effectively. The polyplex was more effective than free siRNA in silencing VEGF in lung cancer cells. The silencing of VEGF was quantified using Western blot and was also reflected in the depletion of HIF-1α levels in the cells treated with the polyplex. VEGF silencing by the polyplex was found to augment the cytotoxic effects of the chemotherapeutic agent 5-fluorouracil. Microarray analysis of the mRNA isolated from cells treated with free siRNA and the polyplex reveal that the VEGF silencing by the polyplex also altered the expression levels of several other genes that have been connected to the proliferation and invasion of lung cancer cells. These results indicate that the PVI complexes can be an effective agent to counter lung cancer.
... 1 Use of viral or nonviral vectors to deliver the therapeutic oligonucleotide to the target cell has been widely explored to overcome the inherent problems associated with administration of the naked oligonucleotide. 2 Majority of gene delivery studies have employed viral vectors due to their superior transfection capabilities. But the high frequency of mutations and packing limitations associated with viral vectors necessitate the search for safer alternates. ...
The present work explores the ability of poly (1-vinyl imidazole) to complex si-RNA against vascular endothelial growth factor (VEGF) and its in vitro efficiency in A549 lung cancer cells. The polyplex formed was found to exhibit 66% complexation efficiency. The complexation was confirmed by gel retardation assay, FTIR and thermal analysis. The blank PVI polymer was not toxic to cells. The polyplex was found to exhibit excellent internalization and escaped the endosome effectively. The polyplex was more effective than the free si-RNA in silencing VEGF in lung cancer cells. The silencing of VEGF was quantified using Western blot which was also reflected in depletion of HIF-1a levels in the cells treated with the polyplex. VEGF silencing by the polyplex was found to augment the cytotoxic effects of the chemotherapeutic agent 5-fluorouracil. Microarray analysis of the mRNA isolated from cells treated with free siRNA and polyplex reveal that the superior VEGF silencing by the polyplex altered the expression levels of several other genes that have been implicated in the proliferation and invasion of lung cancer cells. These results indicate that PVI complexed anti-VEGF can be an effective strategy to counter lung cancer.
... This is used to synthesize other different colloidal nanoparticles made up of organic or hybrid materials. These colloidal nanoparticles have a negative Z-potential, as has been previously reported [41][42][43][44][45][46], and the value of the negative charge depends on the medium in which the colloidal nanoparticles are diluted/dispersed, before the analysis, as well as other components of the pharmaceutical formulations [47][48][49][50][51][52][53][54]. ...
In this study, we investigated the release kinetic of fluorescein from colloidal liquid crystals made from monoglyceride and different non-ionic surfactants. The crystals were physicochemically characterized and the release experiments were carried out under the sink conditions, while mathematical models were described as extrapolations from solutions of the diffusion equation, in different initial and boundary conditions imposed by pharmaceutical formulations. The diffusion equation was solved using Laplace and Fourier transformed functions for release kinetics from infinite reservoirs in a semi-infinite medium. Solutions represents a general square root law and can be applied for the release kinetic of fluorescein from lyotropic colloidal liquid crystals. Akaike, Schwartz, and Imbimbo criteria were used to establish the appropriate mathematical model and the hierarchy of the performances of different models applied to the release experiments. The Fisher statistic test was applied to obtain the significance of differences among mathematical models. Differences of mathematical criteria demonstrated that small or no significant statistic differences were carried out between the various applied models and colloidal formulations. Phenomenological models were preferred over the empirical and semi-empirical ones. The general square root model shows that the diffusion-controlled release of fluorescein is the mathematical models extrapolated for lyotropic colloidal liquid crystals.
Chitosan nanoparticles (NPs) serve as useful multidrug delivery carriers in cancer chemotherapy. Chitosan has considerable potential in drug delivery systems (DDSs) for targeting tumor cells. Doxorubicin (DOX) has limited application due to its resistance and lack of specificity. Chitosan NPs have been used for DOX delivery because of their biocompatibility, biodegradability, drug encapsulation efficiency, and target specificity. In this review, various types of chitosan derivatives are discussed in DDSs to enhance the effectiveness of cancer treatments. Modified chitosan–DOX NP drug deliveries with other compounds also increase the penetration and efficiency of DOX against tumor cells. We also highlight the endogenous stimuli (pH, redox, enzyme) and exogenous stimuli (light, magnetic, ultrasound), and their positive effect on DOX drug delivery via chitosan NPs. Our study sheds light on the importance of chitosan NPs for DOX drug delivery in cancer treatment and may inspire the development of more effective approaches for cancer chemotherapy.
It can be challenging to deliver drugs to cancer cells in a targeted manner at an effective dose. Polymeric nanoparticles (NPs) are promising drug delivery systems that can be targeted to cancer cells using redox responsive elements. More specifically, intracellular and extracellular levels of the antioxidant glutathione (GSH) are elevated in cancer cells and therefore the use of NPs with a cleavable GSH-responsive element allowing these NPs to target cancer cells and trigger the release of their cargo (e.g. anticancer drugs). The aim of this study was to assess the hepatotoxicity of polymeric NP delivery systems with and without a redox sensitive element. Copolymer poly (lactic-co-glycolic acid) (PLGA) and polyethylene glycol (PEG) NPs with (RR-NPs) and without (nRR-NPs) a redox responsive dithiylethanoate ester linker were synthesised and their toxicity assessed in vitro. As the liver is a primary site of NP accumulation, the C3A hepatocyte cell line was used to assess NP toxicity in vitro via investigation of cytotoxicity, cytokine production, genotoxicity, intracellular reactive oxygen species (ROS) production, intracellular calcium concentration, and hepatocyte function (albumin and urea production). The cellular uptake of NPs was also assessed as this may influence the cellular dose and, therefore, the cellular response. Both NPs had no detrimental impact on cell viability. However, both NPs stimulated an increase in cytokine (IL-1ra) and ROS production and decreased hepatocyte function, with the greatest effect observed for nRR-NPs. Only nRR-NPs caused DNA damage. Cells internalised both NPs and caused a (sub-lethal) increase in intracellular calcium levels. Therefore, whilst the NPs did not have a negative impact on cell viability, the NPs were able to elicit sub-lethal toxicity. By using a battery of tests we were able to demonstrate that RR-NPs may be less toxic than nRR-NPs. Our findings can therefore feed into the development of safer and more effective nanomedicines and into the design of testing strategies to assess polymeric NP safety based on knowledge of their mechanism of toxicity.
Lung malignancies accounted for 11% of cancers worldwide in 2020 and remained the leading cause of cancer deaths. About 80% of lung cancers belong to non-small cell lung cancer (NSCLC), which is characterized by extremely high clonal and morphological heterogeneity of tumors and development of multidrug resistance. The improvement of current therapeutic strategies includes several directions. First, increasing knowledge in cancer biology results in better understanding of the mechanisms underlying malignant transformation, alterations in signal transduction, and crosstalk between cancer cells and the tumor microenvironment, including immune cells. In turn, it leads to the discovery of important molecular targets in cancer development, which might be affected pharmaceutically. The second direction focuses on the screening of novel drug candidates, synthetic or from natural sources. Finally, “personalization” of a therapeutic strategy enables maximal damage to the tumor of a patient. The personalization of treatment can be based on the drug screening performed using patient-derived tumor xenografts or in vitro patient-derived cell models. 3D multicellular cancer spheroids, generated from cancer cell lines or tumor-isolated cells, seem to be a helpful tool for the improvement of current NSCLC therapies. Spheroids are used as a tumor-mimicking in vitro model for screening of novel drugs, analysis of intercellular interactions, and oncogenic cell signaling. Moreover, several studies with tumor-derived spheroids suggest this model for the choice of “personalized” therapy. Here we aim to give an overview of the different applications of NSCLC spheroids and discuss the potential contribution of the spheroid model to the development of anticancer strategies.
Bioresponsive nanoparticles (NPs) are of interest for anticancer nanomedicines, owing to the possibility to 'design in' selective modulation of drug release at target sites. Here we describe the double emulsion formulation of redox-responsive NPs based on modified polyethylene glycol (PEG)-co-poly(lactic-co-glycolic acid) (PLGA) block copolymers and oligo (β-aminoesters) (OBAE), both of which contained disulfide linkages, for the co-delivery of a cytotoxic small molecule drug and a nucleic acid. In particular, we focused our attention on docetaxel (DTX) and a siRNA against TUBB3, a gene that encodes for βIII-tubulin, in order to have a synergistic effect in the treatment of lung cancer. Spherical NPs of around 150 nm with negative zeta potential and high loading efficiencies of both drugs were obtained. Stability and release studies showed "on demand" drug release under reducing conditions. Unloaded NPs containing PEG-disulfide-PLGA and OBAE were well-tolerated by lung cancer cells, thus masking the intrinsic cytotoxicity of OBAE, while for intracellular siRNA delivery, redox responsive NPs demonstrated a higher cell internalization with a preferential cytoplasmic accumulation of siRNA, with a subsequent fast gene-silencing efficiency. The viability of cells treated with combined DTX/TUBB3-siRNA NPs significantly decreased as compared to NPs loaded only with DTX, thus showing an efficient combined anticancer effect, due to a substantial reduction of β-tubulin expression. Finally, in an in vivo feasibility study employing an orthotopic lung cancer model, NPs formulated with an anti-luciferase siRNA distributed throughout the lungs following oro-tracheal administration, and demonstrated effective gene knockdown and no apparent cytotoxicity. Taken together, these results show that the double emulsion formulated redox responsive PEG-PLGA and OBAE systems represent a promising new therapeutic approach for the local combined chemo- and gene-therapy of lung cancer.
Liver metastasis (LM) occurs in various cancers, and its early and accurate diagnosis is of great importance. However, the detection of small LMs is still a great challenge because of the subtle differences between normal liver tissue and small metastases. Herein, we prepare glutathione (GSH)-responsive hyaluronic acid-coated iron oxide nanoparticles (HIONPs) for highly sensitive diagnosis of LMs through a facile one-pot method. HIONPs greatly enhance the signal of MRI in tumor metastases as T1 contrast agent (CA), whereas they substantially decrease the signal of liver as T2 CA as they aggregate into clusters upon the high GSH in liver. Consequently, MRI contrasted by HIONPs clearly distinguishes metastatic tumors (bright) from surrounding liver tissues (dark). HIONPs with superior LM contrasting capability and facile synthesis are very promising for clinical translation and indicate a new strategy to develop an ultrasensitive MRI CA for LM diagnosis that exploits high GSH level in the liver.
Nano-drug delivery systems (Nano-DDS) offer powerful advantages in drug delivery and targeted therapy for diseases. Compared to traditional drug formulations, Nano-DDS can increase solubility, biocompatibility, and reduce offtargeted side effects of free drugs. However, they still have some disadvantages that pose a limitation in reaching their full potential in clinical use. Protein adsorption in blood, activation of the complement system, and subsequent sequestration by the mononuclear phagocyte system (MPS) consequently result in nanoparticles (NPs) to be rapidly cleared from the circulation. Therefore, NPs have low drug delivery efficiency. So, it is important to develop stealth NPs for reducing bio– nano interaction. In this review, we first conclude the interaction between NPs and biological environments, such as blood proteins and MPS, and factors influencing each other. Next, we will summarize the new strategies to reduce NPs protein adsorption and uptake by the MPS based on current knowledge of the bio–nano interaction. Further directions will also be highlighted for the development of biomimetic stealth nano-delivery systems by combining targeted strategies for a better therapeutic effect.
Liposomes are promising vectors for pulmonary drug delivery, and have been used in marketed inhalation products. Membrane fluidity is an important property of liposomes. However, the influence of liposome membrane fluidity on its interaction with pulmonary physiological barriers is still unclear and needs elucidation. Here, a series of PEGylated DPPC (1,2-dihexadecanoyl-rac-glycero-3-phosphocholine) liposomes with different membrane fluidity were prepared, and their interaction with different pulmonary physiological barriers, including the mucus permeation capacity, macrophage uptake, trachea distribution and retention behavior, was investigated. The liposomes exhibited sizes of around 100 nm, near-neutral surface charge, and the membrane fluidity increased with increasing cholesterol ratio. In vitro studies showed that the liposomes with lower membrane fluidity presented optimal mucus permeation efficiency, while those with higher membrane fluidity displayed lower macrophage uptake. An in vivo trachea distribution study revealed that liposomes with low or medium membrane fluidity exhibited enhanced trachea permeation. No significant difference in lung retention was found among these liposomes. In conclusion, the mucus permeation and macrophage phagocytosis behavior of liposomes could be well tuned by changing their membrane fluidity.
Lung cancer is the most common cause of cancer-related death in humans worldwide. Various cancer therapies are available to treat cancer but the main causes of concern with most are the toxic effects as well as diagnosis at an advanced stage. These factors lead to high mortality in a majority of patients. These shortcomings can be overcome to a certain extent by either reducing the dose of drug or by early diagnosis. Recently nanotechnology is being explored extensively toward early diagnosis and detection of cancer. Nanotechnology has developed many effective nanomedicines with advance treatment regimens. Various nanosystems have been developed to enhance the therapeutic effect of drugs as well as selective targeting and delivery of drugs to specific sites of tumor. Nanoparticle-based medicines and therapeutics is becoming more important in the treatment of different forms of cancers, especially lung cancer. Detection, diagnosis, imaging, and treatment of lung cancers utilize many nanoparticle-based medicines and therapeutics. Innovations in the application of nanoparticles in lung cancer is a challenging area of research, particularly onsite delivery of drug. This chapter discusses the importance of nanoparticles in the treatment of lung cancer and emerging nanoparticle therapeutics (nanomedicines and drug carriers) and their applications in detection, diagnosis, and cancer therapies.
The size, shape, and underlying chemistries of drug delivery particles are key parameters which govern their ultimate performance in vivo. Responsive particles are desirable for triggered drug delivery, achievable through architecture change and biodegradation to control in vivo fate. Here, polymeric materials are synthesized with linear, hyperbranched, star, and micellar‐like architectures based on 2‐hydroxypropyl methacrylamide (HPMA), and the effects of 3D architecture and redox‐responsive biodegradation on biological transport are investigated. Variations in “stealth” behavior between the materials are quantified in vitro and in vivo, whereby reduction‐responsive hyperbranched polymers most successfully avoid accumulation within the liver, and none of the materials target the spleen or lungs. Functionalization of selected architectures with doxorubicin (DOX) demonstrates enhanced efficacy over the free drug in 2D and 3D in vitro models, and enhanced efficacy in vivo in a highly aggressive orthotopic breast cancer model when dosed over schedules accounting for the biodistribution of the carriers. These data show it is possible to direct materials of the same chemistries into different cellular and physiological regions via modulation of their 3D architectures, and thus the work overall provides valuable new insight into how nanoparticle architecture and programmed degradation can be tailored to elicit specific biological responses for drug delivery.
Chemotherapy, combined with other treatments, is widely applied in the clinical treatment of cancer. However, deficiencies inherited from the traditional route of administration limit its successful application. With the development of nanotechnology, a series of smart nanodelivery systems have been developed to utilize the unique tumor environment (pH changes, different enzymes, and redox potential gradients) and exogenous stimuli (thermal changes, magnetic fields, and light) to improve the curative effect of anticancer drugs. In this review, endogenous and exogenous stimuli are briefly introduced. Among these stimuli, various redox‐sensitive linkages are primarily described in detail, and their application with self‐assembled nanoparticles is recounted. Finally, the application of redox‐responsive self‐assembled nanoparticles in cancer therapy is summarized.
Amphiphilic block co‐polymers composed of poly(ethylene glycol)‐co‐poly(lactide)‐co‐poly(2‐((tert‐butoxycarbonyl)amino)‐3‐propyl carbonate) (PEG‐pLA‐pTBPC) are synthesized in monomer ratios and arrangements to enable assembly into nanoparticles with different sizes and architectures. These materials are based on components in clinical use, or known to be biodegradable, and retain the same fundamental chemistry across “AB” and “BAB” block architectures. In MCF7 and MDA‐MB‐231 breast cancer cells, nanoparticles of <100 nm are internalized most rapidly, by both clathrin‐ and caveolin‐mediated pathways. In THP‐1 cells, polymer architecture and length of the hydrophilic block is the most important factor in the rate of internalization. The organ distributions of systemically injected nanoparticles in healthy mice indicate highest accumulation of the BAB‐blocks in lungs and liver and the lowest accumulation in these organs of a methoxyPEG5000‐pLA‐pTBPC polymer. Conjugation of doxorubicin via a serum‐stable urea linker to the carbonate regions of PEG5000‐pLA‐pTBPC generates self‐assembling nanoparticles which are more cytotoxic in 2D, and penetrate further in 3D spheroids of triple negative breast cancer cells, than the free drug. In an aggressive orthotopic triple negative breast cancer mouse model, the methoxyPEG5000‐pLA‐pTBPC is of similar potency to free doxorubicin but with no evidence of adverse effects in terms of body weight.
Functional ring-opening polymerization (ROP) initiators can instill a wide array of chemical, physical, and biological effects into a polymeric chain. Highlighting the versatility of this "active" initiator approach, a broad range of characteristics can be achieved through the use of initiators with chemistries spanning from drugs and dyes (key in the case of drug delivery or nanoparticle applications) through to radically active monomers, polymerization transfer agents, and catalysts. The selection of a suitable "active" initiator (monomers for tandem reactions, dyes, drugs, stereo-catalysts, etc.) can not only provide the final polymers with interesting application potential but also facilitate the implementation of ROP reactions in tandem with other polymerization techniques. Overall, this review will highlight that functionalities and properties can be effectively tuned by exploiting simple chemistry approaches, allowing readers to identify how these approaches could be of benefit to their own work in a range of applications including drug/gene delivery, amphiphilic bio/de-gradable carriers, drug/scent controlled release, and stereo-controlled polymers.
The main reason for limited efficacy of anticancer drug is the poor accretion of administered amount of drug within the tumor. Here, chitosan folate capped dual responsive mesoporous silica nanoparticles (MSNs) which can actively target cancer cells, and provide burst release of loaded anticancer drug within tumor cells and ultimately leading to improved therapeutic efficacy were synthesized. MSNs were synthesized using most economic silica source, sodium silicate. Doxorubicin (DOX) was loaded within the pores of MSNs and these drug loaded MSNs were first reacted with cystamine dihydrochloride followed by capping with chitosan-folate conjugate (CH-FA) to produce dual (redox and pH) responsive nanoparticles with the ability to actively target breast cancer cells. A triggered release of DOX from MSNs under acidic redox (pH 5.5, 10mM GSH) environment was confirmed by in vitro release studies. The formulation exhibited 2.14 and 1.65 folds higher cytotoxicity than free drug against MCF-7 and MDA-MB-231 cells. DOX-MSN-SS-CH-FA showed superior tumor suppressing activity as compared to DOX-MSN or DOX alone in the treatment of Ehrlich Ascites Carcinoma (EAC) induced breast cancer with significantly reduced hematological and organ specific toxicities associated with DOX treatment.
Triple negative or basal-like breast cancer (TNBC) is characterised by aggressive progression, lack of standard therapies and poorer overall survival rates for patients. The bad prognosis, high rate of relapse and resistance against anticancer drugs have been associated with a highly abnormal loss of redox control in TNBC cells. Here, we developed docetaxel (DTX)-loaded micellar-like nanoparticles (MLNPs), designed to address the aberrant TNBC biology through the placement of redox responsive cross-links designed into a terpolymer. The MLNPs were derived from poly(ethyleneglycol)-b-poly(lactide)-co-poly(N3-α-ε-caprolactone) with a disulfide linker pendant from the caprolactone regions in order to cross-link adjacent chains. The terpolymer contained both polylactide and polycaprolactone to provide a balance of accessibility to reductive agents necessary to ensure stability in transit, but rapid micellar breakdown and concomitant drug release, when in breast cancer cells with increased levels of reducing agents. The empty micellar-like nanoparticles did not show any cytotoxicity in vitro in 2D monolayers of MDA-MB-231 (triple-negative breast cancer), MCF7 (breast cancer) and MCF10A (normal breast epithelial cell line), whereas DTX-loaded reducible crosslinked MLNPs exhibited higher cytotoxicity against TNBC and breast cancer cells which present high intracellular levels of glutathione. Crosslinked and non-crosslinked MLNPs showed high and concentration-dependent cellular uptake in monolayers and tumour spheroids, including when assessed in co-cultures of TNBC cells and cancer-associated fibroblasts. DTX loaded crosslinked MLNPs showed the highest efficacy against 3D spheroids of TNBC, in addition the MLNPs also induced higher levels of apoptosis, as assessed by annexin V/PI assays and increased caspase 3/7 activity in MDA-MB-231 cells in comparison to cells treated with DTX-loaded un-crosslinked MLNP (used as a control) and free DTX. Taken together these data demonstrate that the terpolymer micellar-like nanoparticles with reducible crosslinks have high efficacy in both 2D and 3D in vitro cancer models by targeting the aberrant biology, i.e. loss of redox control of this type of tumour, thus may be promising and effective carrier systems for future clinical applications in TNBC.
Poly(lactic-co-glycolic acid) (PLGA) is a versatile synthetic copolymer that is widely used in pharmaceutical applications. This is because it is well-tolerated in the body, and copolymers of varying physicochemical properties are readily available via ring-opening polymerization. However, native PLGA polymers are hard to track as drug delivery carriers when delivered to subcellular spaces, due to the absence of an easily accessible “handle” for fluorescent labeling. Here we show a one-step, scalable, solvent-free, synthetic route to fluorescent blue (2-aminoanthracene), green (5-aminofluorescein), and red (rhodamine-6G) PLGA, in which every polymer chain in the sample is fluorescently labeled. The utility of initiator-labeled PLGA was demonstrated through the preparation of nanoparticles, capable of therapeutic subcellular delivery to T-helper-precursor-1 (THP-1) macrophages, a model cell line for determining in vitro biocompatibility and particle uptake. Super resolution confocal fluorescence microscopy imaging showed that dye-initiated PLGA nanoparticles were internalized to punctate regions and retained bright fluorescence over at least 24 h. In comparison, PLGA nanoparticles with 5-aminofluorescein introduced by conventional nanoprecipitation/encapsulation showed diffuse and much lower fluorescence intensity in the same cells and over the same time periods. The utility of this approach for in vitro drug delivery experiments was demonstrated through the concurrent imaging of the fluorescent drug doxorubicin (λex = 480 nm, λem = 590 nm) with carrier 5-aminofluorescein PLGA, also in THP-1 cells, in which the intracellular locations of the drug and the polymer could be clearly visualized. Finally, the dye-labeled particles were evaluated in an in vivo model, via delivery to the nematode Caenorhabditis elegans, with bright fluorescence again apparent in the internal tract after 3 h. The results presented in this manuscript highlight the ease of synthesis of highly fluorescent PLGA, which could be used to augment tracking of future therapeutics and accelerate in vitro and in vivo characterization of delivery systems prior to clinical translation.
Main principles of applicability of polymers for biomedical purposes with the emphasis on their physico‐chemical properties and solution behavior are reviewed. The influence of molecular weight of the polymers and their degradability are also discussed. Thermosensitive polymers are pointed out including additional effects such as hydrogen bonds and vitrification. Several examples of polymers currently used or under development are presented and their advantages and drawbacks are analyzed.
It remains a challenge to increase drug tumor-specific accumulation as well as to achieve intracellular-controlled drug release for hepatocellular carcinoma (HCC) chemotherapy. Herein, we developed a dual-functional biodegradable micellar system constituted by glycyrrhetinic acid coupling poly(ethylene glycol)-disulfide linkage-poly(lactic-co-glycolic acid) (GA-PEG-SS-PLGA) to achieve both hepatoma-targeting and redox-responsive intracellular drug release. Tanshinone IIA (TAN IIA), an effective anti-HCC drug, was encapsulated. Notably, it exhibited rapid aggregation and faster drug release in 10 mM dithiothreitol compared with the redox-insensitive control. Furthermore, GA-decorated micelles revealed HCC-specific cellular uptake in human liver cancer HepG2 cells with an energy-dependent manner, in which micropinocytosis and caveolae-mediated endocytosis were demonstrated as the major cellular pathways. The enhanced cytotoxicity and pro-apoptotic effects against HepG2 cells in vitro were observed, mediated by up-regulation of the intracellular ROS level, the increased cell cycle arrest at S phase, enhanced necrocytosis and up-regulation of caspase 3/7, P38 protein expression. In addition, TAN IIA-loaded micelles had a significantly prolonged circulation time, improved bioavailability, and resulted in an increased accumulation of TAN IIA in the liver. With the synergistic effects of HCC-targeting and controlled drug release, TAN IIA-loaded GA-PEG-SS-PLGA micelles significantly inhibited tumor growth and increased survival time in a mouse HCC-xenograft model. Collectively, the GA-PEG-SS-PLGA micelles with HCC-targeting and redox-sensitive characters would provide a novel strategy to deliver TAN IIA effectively for HCC therapy.
Local administration of therapeutics by inhalation for treatment of lung diseases has the ability to deliver drugs, nucleic acids and peptides specifically to the site of their action and therefore enhance the efficacy of the treatment, limit the penetration of nebulized therapeutic agent(s) into the bloodstream and consequently decrease adverse systemic side effects of the treatment. Nanotechnology allows for a further enhancement of the treatment efficiency. The present review analyzes modern therapeutic approaches of inhaled nanoscale-based pharmaceutics for the detection and treatment of various lung diseases.
Copyright © 2015. Published by Elsevier B.V.
Nanoparticles are extensively studied for drug delivery and are proving to be effective in drug delivery and the diagnostic field. Drug delivery to lungs has its advantages over other routes of administration. Inhalable powders consisting of nanoparticles is gaining much interest in respiratory research and clinical therapy. Particle engineering technique is a key factor to develop inhalable formulations that can successfully deliver drug with improved therapeutic effect and enhanced targeting. Inhalable nanoparticles in the solid-state dry powders for targeted pulmonary delivery offers unique advantages and is an exciting new area of research. Nasal delivery of inhalable nanoparticulate powders is gaining research attention recently, particularly in vaccine applications, systemic drug delivery in the treatment of pain, and non-invasive brain targeting. Fundamental aspects and recent advancements along with future prospects of inhalable powders consisting of nanoparticles in the solid-state for respiratory delivery are presented.
Copyright © 2015. Published by Elsevier Inc.
Pulmonary drug delivery represents the best way of treating lung diseases, since it allows direct delivery of the drug to the site of action, with few systemic effects. Meanwhile, the lungs may be used as a portal of entry to the body, allowing systemic delivery of drugs via the airway surfaces into the bloodstream. In both cases, the therapeutic effect of the inhaled drug can be optimized by embedding in appropriately engineered inhalable carriers, which can protect the drug against lung defense mechanisms and promote drug transport across the extracellular and cellular barriers. To this purpose, the attention has been very recently focused on polymeric nanoparticles (NPs). The aim of this review is to offer an overview on the recent advances in NPs for pulmonary drug delivery. After a description of the main challenges encountered in developing novel inhaled products, the design rules to engineer polymeric NPs for inhalation, and in so doing to overcome barriers imposed by the lungs anatomy and physiology, are described. Then, the state-of-art on inhalable biocompatible polymeric NPs based on enzymatically-degradable natural polymers and biodegradable poly(ester)s is presented, with a special focus on NP-based dry powders for inhalation. Finally, the in vitro/in vivo models useful to address the never-ending toxicological debate related to the use of NPs for inhalation are described.
The clinical efficacy of cytotoxic drugs in the treatment of cancer is often hampered by poor pharmacodynamics and systemic toxicity. Here, we describe the design and synthesis of a new PEG-based system for the delivery of the cytotoxic camptothecin (CPT) into tumor cells that overexpress luteinizing hormone releasing hormone receptor (LHRHR). A novel functional reducible camptothecin (CPT) block copolymer conjugate was prepared using atom transfer radical polymerization (ATRP). The use of ATRP in the design and synthesis of the copolymer prodrug facilitated high drug loading and specific delivery to tumor cells. The efficacy of the polymer conjugate was evaluated in appropriate cancer cell lines in vitro. Cytotoxic potency was comparable to that of free CPT in LHRHR positive cell lines after 72 hours, whereas little cytotoxicity was observed in LHRHR negative lines. The study also evaluated the effects of polymer-based therapeutics on human peripheral blood mononuclear cells (PBMC). Free CPT demonstrated indiscriminate toxicity against the immune cells, with impairment of PBMC proliferation and a reduction in CD8+, CD4+ T cell populations. The camptothecin (CPT) block copolymer demonstrated a significant improvement in cell proliferation and maintenance of CD8+ cells.
This literature review is a compilation of the composition and, in most cases, the preparation instructions for simulated biological fluids that may be used as dissolution media in the evaluation of dissolution profiles and amount of drug released from pharmaceutical dosage forms. The use of simulated biological fluids can give a better understanding of the release mechanisms and possible in vivo behavior of a product and enhance the predictive capability of the dissolution testing. A summary of the major characteristics of the most used routes of administration that may affect dissolution and absorption of drug substances is presented. The routes and simulated biological fluids covered by this review are: • Parenteral: simulated body fluid and simulated synovial fluid. • Oral: fasted-state simulated gastric fluid, fed-state simulated gastric fluid, fasted state simulated intestinal fluid, fed-state simulated intestinal fluid, simulated colonic fluid, fasted state simulated colonic fluid, and fed-state simulated colonic fluid. • Buccal and sublingual: simulated saliva. • Pulmonary: simulated lung fluid. • Vaginal: simulated vaginal fluid and simulated semen. • Ophthalmic: simulated tears. Simulated sweat is also included. Some examples of how these simulated biological fluids are used to evaluate dosage forms are included in each route of administration.
Several PEGylated polyester-based nanoncologicals have been proposed in the literature, some of them nowadays being under preclinical/clinical trials or marketed. In this review, we describe the main features of PEGylated polyesters and their correspondent nanocarriers. A first part is devoted to intravenously injectable PEGylated nanocarriers, which represent the systems most investigated so far. After describing fundamental design rules dictated by the administration route, PEGylated nanocarriers currently under preclinical/clinical investigation or in the market will be described from a technological point of view and related therapeutic implications discussed. Finally, new perspective of use of PEGylated nanocarriers for oral and pulmonary delivery of anticancer drugs will be considered.
Spurred by recent progress in materials chemistry and drug delivery, stimuli-responsive devices that deliver a drug in spatial-, temporal- and dosage-controlled fashions have become possible. Implementation of such devices requires the use of biocompatible materials that are susceptible to a specific physical incitement or that, in response to a specific stimulus, undergo a protonation, a hydrolytic cleavage or a (supra)molecular conformational change. In this Review, we discuss recent advances in the design of nanoscale stimuli-responsive systems that are able to control drug biodistribution in response to specific stimuli, either exogenous (variations in temperature, magnetic field, ultrasound intensity, light or electric pulses) or endogenous (changes in pH, enzyme concentration or redox gradients).
Regional chemotherapy was first used for lung cancer 30 years ago. Since then, new methods of drug delivery and pharmaceuticals have been investigated in vitro, and in animals and humans. An extensive review of drug delivery systems, pharmaceuticals, patient monitoring, methods of enhancing inhaled drug deposition, safety and efficacy, and also additional applications of inhaled chemotherapy and its advantages and disadvantages are presented. Regional chemotherapy to the lung parenchyma for lung cancer is feasible and efficient. Safety depends on the chemotherapy agent delivered to the lungs and is dose-dependent and time-dependent. Further evaluation is needed to provide data regarding early lung cancer stages, and whether regional chemotherapy can be used as neoadjuvant or adjuvant treatment. Finally, inhaled chemotherapy could one day be administered at home with fewer systemic adverse effects.
In past two decades poly lactic-co-glycolic acid (PLGA) has been among the most attractive polymeric candidates used to fabricate devices for drug delivery and tissue engineering applications. PLGA is biocompatible and biodegradable, exhibits a wide range of erosion times, has tunable mechanical properties and most importantly, is a FDA approved polymer. In particular, PLGA has been extensively studied for the development of devices for controlled delivery of small molecule drugs, proteins and other macromolecules in commercial use and in research. This manuscript describes the various fabrication techniques for these devices and the factors affecting their degradation and drug release.
To develop a novel brain drug delivery system based on self-assembled poly(ethyleneglycol)-poly (D,L-lactic-co-glycolic acid) (PEG-PLGA) polymersomes conjugated with lactoferrin (Lf-POS). The brain delivery properties of Lf-POS were investigated and optimized.
Three formulations of Lf-POS, with different densities of lactoferrin on the surface of polymersomes, were prepared and characterized. The brain delivery properties in mice were investigated using 6-coumarin as a fluorescent probe loaded in Lf-POS (6-coumarin-Lf-POS). A neuroprotective peptide, S14G-humanin, was incorporated into Lf-POS (SHN-Lf-POS); a protective effect on the hippocampuses of rats treated by Amyloid-β(25-35) was investigated by immunohistochemical analysis.
The results of brain delivery in mice demonstrated that the optimized number of lactoferrin conjugated per polymersome was 101. This obtains the greatest blood-brain barrier (BBB) permeability surface area(PS) product and percentage of injected dose per gram brain (%ID/g brain). Immunohistochemistry revealed the SHN-Lf-POS had a protective effect on neurons of rats by attenuating the expression of Bax and caspase-3 positive cells. Meanwhile, the activity of choline acetyltransferase (ChAT) had been increased compared with negative controls.
These results suggest that lactoferrin functionalized self-assembled PEG-PLGA polymersomes could be a promising brain-targeting peptide drug delivery system via intravenous administration.
Micelles are colloidal particles with a size around 5-100 nm which are currently under investigation as carriers for hydrophobic drugs in anticancer therapy. Currently, five micellar formulations for anticancer therapy are under clinical evaluation, of which Genexol-PM has been FDA approved for use in patients with breast cancer. Micelle-based drug delivery, however, can be improved in different ways. Targeting ligands can be attached to the micelles which specifically recognize and bind to receptors overexpressed in tumor cells, and chelation or incorporation of imaging moieties enables tracking micelles in vivo for biodistribution studies. Moreover, pH-, thermo-, ultrasound-, or light-sensitive block copolymers allow for controlled micelle dissociation and triggered drug release. The combination of these approaches will further improve specificity and efficacy of micelle-based drug delivery and brings the development of a 'magic bullet' a major step forward.
We developed polylysine based DNA nanoparticles (DNA NPs) that contain disulfide linkage in the carrier and demonstrated that this reducible DNA NP enhances in vitro gene transfer via an extracellular mechanism. Polylysine was conjugated through an N-terminal cysteine to a polyethylene glycol chain (PEG) by either a disulfide bond (SS) or a thioether bond (CS), and the resulting PEG-peptide conjugates were used to compact plasmid DNA into reducible SS-DNA NPs or non-reducible CS-DNA NPs with identical physical properties. SS-DNA NPs mediated more than 10-fold higher in vitro gene transfer. Others have suggested that disulfide bonds in synthetic gene carriers undergo cleavage in the reducing environment inside the cell, allowing increased intracellular DNA release. In this study, however, both higher cellular uptake of SS-DNA NPs and inhibition of SS-DNA NP mediated in vitro gene transfer by blocking extracellular free thiols suggested an extracellular mechanism. DePEGylation of SS-DNA NPs by extracellular thiols caused aggregation which might lead to higher cellular uptake and higher transgene expression. A series of SS-DNA NPs prepared with stabilized disulfide bonds survived the extracellular environment without aggregation but lost the superior gene transfer ability, indicating that, in our system, intracellular mechanisms are not involved. These results provided further insight into the mechanisms of in vitro gene transfer enhancement by introducing reducible linkages, contributing to the rational design of more efficient non-viral gene delivery systems.
Försters resonance energy transfer (FRET) microscopy is widely used for the analysis of protein interactions in intact cells. However, FRET microscopy is technically challenging and does not allow assessing interactions in large cell numbers. To overcome these limitations we developed a flow cytometry-based FRET assay and analysed interactions of human and simian immunodeficiency virus (HIV and SIV) Nef and Vpu proteins with cellular factors, as well as HIV Rev multimer-formation.
Amongst others, we characterize the interaction of Vpu with CD317 (also termed Bst-2 or tetherin), a host restriction factor that inhibits HIV release from infected cells and demonstrate that the direct binding of both is mediated by the Vpu membrane-spanning region. Furthermore, we adapted our assay to allow the identification of novel protein interaction partners in a high-throughput format.
The presented combination of FRET and FACS offers the precious possibility to discover and define protein interactions in living cells and is expected to contribute to the identification of novel therapeutic targets for treatment of human diseases.
Whereas there is biological evidence that the reductive cleavage of disulfide bonds is critical for the activation of endocytosed macromolecules such as toxins, immunotoxins, and other drug carriers, virtually nothing is known about the specifics of this cleavage. To study this process, a model compound was synthesized in which a radioiodinated tyramine was linked through a disulfide bond to an undegradable carrier, poly(D-lysine), known to be efficiently endocytosed. Cultured Chinese hamster ovary cells were pulse-labeled with this probe, and the disulfide cleavage was measured as released acid-soluble radioactivity at different times of chase. Pulse-labeled cells were also subjected to subcellular fractionation to identify intracellular structures associated with disulfide cleavage. Cleavage began without lag, amounted to about approximately 7% of the initial cell-bound radioactivity in the first hour and continued for more than 6 h. It was abolished in the presence of N-ethylmaleimide. When sulfhydryl groups present at the cell surface were blocked with cell-impermeant sulfhydryl reagent, the initial phase of disulfide cleavage was inhibited, indicating that cleavage began at the cell surface. A long-lasting intracellular phase of disulfide cleavage began after about approximately 30 min of chase. Subcellular fractionation and kinetic analysis indicated that neither lysosomes nor endosomes were participating in that phase, leaving the Golgi apparatus as the most probable site of endocytic disulfide cleavage.
Evidence had been provided that a disulfide-linked [125I]iodotyramine/poly(D-lysine) conjugate was reductively cleaved when bound nonspecifically to the surface of Chinese hamster ovary (CHO) cells and that this cleavage was abolished by membrane-impermeant sulfhydryl blockers. The same blockers were subsequently found to inhibit the cytotoxicity of diphtheria toxin, a disulfide-linked heterodimer that binds to a specific surface receptor and must undergo chain separation to exert its cytotoxicity. This suggested that the disulfides of both macromolecules might be cleaved by a thiol-disulfide interchange reaction, possibly mediated by protein disulfide-isomerase (PDI, EC 5.3.4.1). We tested whether inhibitors of PDI--in particular, bacitracin and anti-PDI antibodies--might mimic the two effects of sulfhydryl blockers. Both bacitracin and anti-PDI antibodies were effective in inhibiting both reductive processes. This strongly suggests that the disulfide cleavage in the two membrane-bound macromolecules is mediated by PDI and that this enzyme, besides its known retention in the endoplasmic reticulum, must also be exposed at the plasma membrane. This paper points to other potentially important disulfide reductions that might be catalyzed by surface-associated PDI. It thereby broadens the known functions of an enzyme already known for its multifunctional properties.
Remarkably, with the exception of anaesthetic gases, the ancient human practice of inhaling substances into the lungs for systemic effect has only just begun to be adopted by modern medicine. Treatment of asthma by inhaled drugs began in earnest in the 1950s, and now such 'topical' or targeted treatment with inhaled drugs is considered for treating many other lung diseases. More recently, major advances have led to increasing interest in systemic delivery of drugs by inhalation. Small molecules can be delivered with very rapid action, low metabolism and high bioavailability; and macromolecules can be delivered without injections, as highlighted by the recent approval of the first inhaled insulin product. Here, we review these advances, and discuss aspects of lung physiology and formulation composition that influence the systemic delivery of inhaled therapeutics.
Polymeric materials that respond to a variety of endogenous and external stimuli are actively developed to overcome the main barriers to successful systemic delivery of therapeutic nucleic acids. Here, an overview of viable stimuli that are proved to improve systemic delivery of nucleic acids is provided. The main focus is placed on nucleic acid delivery systems (NADS) based on polymers that respond to pathological or physiological changes in pH, redox state, enzyme levels, hypoxia, and reactive oxygen species levels. Additional discussion is focused on NADS suitable for applications that use external stimuli, such as light, ultrasound, and local hyperthermia.
Stimuli-responsive polymeric nanosystems that can respond to biological stimuli such as pH, temperature, glucose, enzymes or redox conditions have been extensively explored for different biomedical applications. Among these, redox conditions should be the most useful stimulus in biological systems, which rely on the significantly different redox states in the circulation/extracellular fluids and intracellular compartments. By incorporation of redox-responsive linkages such as disulfide and diselenide into polymers, different redox-responsive polymeric nanosystems can be fabricated. In this review article, a number of redox-responsive polymeric therapeutic nanosystems and their design principles are included. Recent advances in these redox-responsive polymeric therapeutic nanosystems for controlled cytoplasmic delivery of a number of bioactive molecules (e.g. drugs, biological proteins, plasmid DNA, siRNA) are also highlighted. This review will provide useful information for the design and biomedical applications of redox-responsive polymeric therapeutic nanosystems, which will attract great research interest from scientists in chemistry, materials, biology, medicine and interdisciplinary areas.
One of the most challenging and clinically important goals in nanomedicine is to deliver imaging and therapeutic agents to solid tumors. Here we discuss the recent design and development of stimuli-responsive smart nanoparticles for targeting the common attributes of solid tumors such as their acidic and hypoxic microenvironments. This class of stimuli-responsive nanoparticles is inactive during blood circulation and under normal physiological conditions, but is activated by acidic pH, enzymatic up-regulation, or hypoxia once they extravasate into the tumor microenvironment. The nanoparticles are often designed to first "navigate" the body's vascular system, "dock" at the tumor sites, and then "activate" for action inside the tumor interstitial space. They combine the favorable biodistribution and pharmacokinetic properties of nanodelivery vehicles and the rapid diffusion and penetration properties of smaller drug cargos. By targeting the broad tumor habitats rather than tumor-specific receptors, this strategy has the potential to overcome the tumor heterogeneity problem and could be used to design diagnostic and therapeutic nanoparticles for a broad range of solid tumors.
Copyright © 2015. Published by Elsevier B.V.
Achieving sustained drug delivery to mucosal surfaces is a major challenge due to the presence of the protective mucus layer that serves to trap and rapidly remove foreign particulates. Nanoparticles engineered to rapidly penetrate mucosal barriers (mucus-penetrating particles, "MPP") have shown promise for improving drug distribution, retention and efficacy at mucosal surfaces. MPP are densely coated with polyethylene glycol (PEG), which shields the nanoparticle core from adhesive interactions with mucus. However, the PEG density required to impart the "stealth" properties to nanoparticles in mucus, and thus, uniform distribution in vivo, is still unknown. We prepared biodegradable poly(lactic-co-glycolic acid) (PLGA) nanoparticles with a range of PEG surface densities by blending various ratios of a diblock copolymer of PLGA and 5 kDa poly(ethylene glycol) (PLGA-PEG5k) with PLGA. We then evaluated the impact of PEG surface density, measured using an 1H NMR method, on mucin binding in vitro, nanoparticle transport in freshly obtained human cervicovaginal mucus (CVM) ex vivo, and nanoparticle distribution in the mouse cervicovaginal tract in vivo. We found that at least 5% PEG was required to effectively shield the nanoparticle core from interacting with mucus components in vitro and ex vivo, thus leading to enhanced nanoparticle distribution throughout the mouse vagina in vivo. We then demonstrated that biodegradable MPP could be formulated from blends of PLGA and PLGA-PEG polymers of various molecular weights, and that these MPP provide tunable drug loading and drug release rates and durations. Overall, we describe a methodology for rationally designing biodegradable, drug-loaded MPP for more uniform delivery to the vagina.
Significance
Therapeutically relevant lung gene therapy is yet to be achieved. We introduce a highly translatable gene delivery platform for inhaled gene therapy based on state-of-the-art biodegradable polymers, poly(β-amino esters). The newly designed system is capable of overcoming challenging biological barriers, thereby providing robust transgene expression throughout the entire luminal surface of mouse lungs. Moreover, it provides markedly greater overall transgene expression in vivo compared with gold standard platforms, including a clinically tested system. The clinical relevance is further underscored by the excellent safety profile as well as long-term and consistent transgene expression achieved following a single and repeated administrations, respectively.
Here we have investigated the effect of enshrouding polymer-coated nanoparticles (NPs) with polyethylene glycol (PEG) on the adsorption of proteins and uptake by cultured cells. PEG was covalently linked to the polymer surface to the maximal grafting density achievable under our experimental conditions. Changes in the effective hydrodynamic radius of the NPs upon adsorption of human serum albumin (HSA) and fibrinogen (FIB) were measured in situ by using fluorescence correlation spectroscopy (FCS). For NPs without a PEG shell, a thickness increase of around 3 nm, corresponding to HSA monolayer adsorption, was measured at high HSA concentration. Only 50% of this value was found for NPs with PEGylated surfaces. While the size increase clearly reveals formation of a protein corona also for PEGylated NPs, fluorescence lifetime measurements and quenching experiments suggest that the adsorbed HSA molecules are buried within the PEG shell. For FIB adsorption onto PEGylated NPs, even less change in NP diameter was observed. In vitro uptake of the NPs by 3T3 fibroblasts was reduced to around 10% upon PEGylation with PEG chains of 10 kDa. Thus, even though the PEG coatings did not completely prevent protein adsorption, the PEGylated NPs still displayed a pronounced reduction of cellular uptake with respect to bare NPs, which is to be expected if the adsorbed proteins are not exposed on the NP surface.
The use of nanoparticulate pharmaceutical drug delivery systems (NDDSs) to enhance the in vivo effectiveness of drugs is now well established. The development of multifunctional and stimulus-sensitive NDDSs is an active area of current research. Such NDDSs can have long circulation times, target the site of the disease and enhance the intracellular delivery of a drug. This type of NDDS can also respond to local stimuli that are characteristic of the pathological site by, for example, releasing an entrapped drug or shedding a protective coating, thus facilitating the interaction between drug-loaded nanocarriers and target cells or tissues. In addition, imaging contrast moieties can be attached to these carriers to track their real-time biodistribution and accumulation in target cells or tissues. Here, I highlight recent developments with multifunctional and stimuli-sensitive NDDSs and their therapeutic potential for diseases including cancer, cardiovascular diseases and infectious diseases.
The release of azidothymidine from macromolecular prodrugs was designed to respond to the intracellular disulfide reshuffling. This drug has no thiol groups, and a response to this trigger was engineered using a self-immolative linker. The resulting formulations were fast-acting, efficacious, and highly potent with regards to suppressing the infectivity of the virus.
Research in pulmonary drug delivery has focused mainly on new particle or device technologies to improve the aerosol generation and pulmonary deposition of inhaled drugs. Although substantial progress has been made in this respect, no significant advances have been made that would lead pulmonary drug delivery beyond the treatment of some respiratory diseases. One main reason for this stagnation is the still very scarce knowledge about the fate of inhaled drug or carrier particles after deposition in the lungs. Improvement of the aerosol component alone is no longer sufficient for therapeutic success of inhalation drugs; a paradigm shift is needed, with an increased focus on the pulmonary barriers to drug delivery. In this Review, we discuss some pathophysiological disorders that could benefit from better control of the processes after aerosol deposition, and pharmaceutical approaches to achieve improved absorption across the alveolar epithelium, prolonged pulmonary clearance, and targeted delivery to specific cells or tissues.
Multidentate ligands are expected to improve the performance of inorganic nanoparticles (NPs) in biological application. Designing robust multidentate ligands by a facile way and understanding the impact of ligand composition on NP's property are greatly important. We report the effective synthesis of hydrophilic copolymers containing pendent thiol groups along a polyethylene glycol (PEG) methacrylate backbone by classical free radical copolymerization. Gold nanoparticles (AuNPs) coated by these multidentate ligands with two different ratios of thiols to PEG segment (∼1:1 and 1:2) showed much higher colloidal stability in the presence of dithiothreitol (DTT) than AuNPs coated by monothiol-anchored PEG, and AuNPs coated by ligands with higher fraction of thiol groups showed slightly better resistance to DTT competition than did AuNPs coated by ligands with lower thiol fraction, but both of them exhibited excellent stabilities in biological media without obvious difference. In vitro study of uptake by macrophages did not showed significant difference between the two AuNPs with very low endocytosis. However, AuNPs coated by ligands with higher PEG content were found to accumulate in liver with a significantly lower level but a higher level in spleen than AuNPs coated by ligands with lower PEG contents. Moreover, the AuNPs coated with by ligands with higher PEG content showed higher tumor uptake. Additionally, AuNPs coated with both ligands demonstrated good biocompatibility as evaluated by cytotoxicity assays and histological analysis. Together, the composition of multidentate ligands will not only affect the stability of NPs under extreme conditions but also result in quite different fate of NPs in vivo, which can be tailored case by case.
Although nanocarriers hold promise for cancer chemotherapy, their intracellular drug delivery pathways are not fully understood. In particular, the influence of nanocarrier stability on cellular uptake is still uncertain. By physically loading hydrophobic FRET probes, we revealed different intracellular drug delivery routes of self-assembled and disulfide bonded micelles. The self-assembled micelles were structurally dissociated by micelle-membrane interactions, and the hydrophobic probes were distributed on the plasma membrane. Alternatively, intact disulfide bonded micelles carrying hydrophobic probes were internalized into cancer cells via multiple endocytic pathways. Following internalization, disulfide bonded micelles were decomposed in early endosomes by glutathione-mediated disulfide bond reduction, exposing the probes to intracellular organelles.
About six years ago, almost simultaneously, three independent groups reported the synthesis of a novel class of dendritic molecules. These molecules were equipped with a focal trigger; reaction at the trigger initiated the fragmentation of the dendrimer to its building blocks in a domino-like manner with consequent release of at least two tail-units. We termed these molecules “self-immolative dendrimers” and demonstrated their distinctive ability to achieve molecular amplification of the initial triggering signal. The unique amplification effect obtained by self-immolative dendrimers is useful in the fields of drug delivery systems and diagnostic applications. This highlight summarizes the design, function, and applications of this unique class of molecules.
Two poly(ethylene glycol) (PEG)-peptides were synthesized and tested for their ability to bind to plasmid DNA and form soluble DNA condensates with reduced spontaneous gene expression. PEG-vinyl sulfone or PEG-orthopyridyl disulfide were reacted with the sulfhydryl of Cys-Trp-Lys18 (CWK18) resulting in the formation of nonreducible (PEG-VS-CWK18) and reducible (PEG-SS-CWK18) PEG- peptides. Both PEG-peptides were prepared on a micromole scale, purified by RP-HPLC in >80% yield, and characterized by 1H NMR and MALDI-TOF. PEG-peptides bound to plasmid DNA with an apparent affinity that was equivalent to alkylated (Alk)CWK18, resulting in DNA condensates with a mean diameter of 80–90 nm and Z (zeta) potential of +10 mV. The particle size of PEG-peptide DNA condensates was constant throughout the DNA concentration range of 0.05–2 mg/mL, indicating these to be approximately 20-fold more soluble than AlkCWK18 DNA condensates. The spontaneous gene transfer to HepG2 cells mediated by PEG-VS-CWK18 DNA conden- sates was over two orders of magnitude lower than PEG-SS-CWK18 DNA condensates and three orders of magnitude lower than AlkCWK18 DNA condensates. PEG-VS-CWK18 efficiently blocked in vitro gene transfer by reducing cell uptake. The results indicate that a high loading density of PEG on DNA is necessary to achieve highly soluble DNA condensates that reduce spontaneous in vitro gene transfer by blocking nonspecific uptake by HepG2 cells. These two properties are important for developing targeted gene delivery systems to be used in vivo.
Highly compacted DNA nanoparticles, composed of single molecules of plasmid DNA compacted with block copolymers of poly-l-lysine and 10kDa polyethylene glycol (CK(30)PEG(10k)), mediate effective gene delivery to the brain, eyes and lungs in vivo. Nevertheless, we found that CK(30)PEG(10k) DNA nanoparticles are immobilized by mucoadhesive interactions in sputum that lines the lung airways of patients with cystic fibrosis (CF), which would presumably preclude the efficient delivery of cargo DNA to the underlying epithelium. We previously found that nanoparticles can rapidly penetrate human mucus secretions if they are densely coated with low MW PEG (2-5kDa), whereas nanoparticles with 10kDa PEG coatings were immobilized. We thus sought to reduce mucoadhesion of DNA nanoparticles by producing CK(30)PEG DNA nanoparticles with low MW PEG coatings. We examined the morphology, colloidal stability, nuclease resistance, diffusion in human sputum and in vivo gene transfer of CK(30)PEG DNA nanoparticles prepared using various PEG MWs. CK(30)PEG(10k) and CK(30)PEG(5k) formulations did not aggregate in saline, provided partial protection against DNase I digestion and exhibited the highest gene transfer to lung airways following inhalation in BALB/c mice. However, all DNA nanoparticle formulations were immobilized in freshly expectorated human CF sputum, likely due to inadequate PEG surface coverage.
Although few experimental studies have been handled so far to exploit the potential of poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) in the production of dry powders for antibiotic inhalation, there has been no comprehensive study on the role played by NP composition. In this work, we try to shed light on this aspect by designing and developing a pulmonary delivery system for antibiotics, such as tobramycin (Tb), based on PLGA NPs embedded in an inert microcarrier made of lactose, referred to as nano-embedded micro-particles (NEM). At nanosize level, helper hydrophilic polymers were used to impart the desired surface, bulk and release properties to PLGA NPs prepared by a modified emulsion-solvent diffusion technique. Results showed that poly(vinyl alcohol) (PVA) and chitosan (CS) are essential to optimise the size and modulate the surface properties of Tb-loaded PLGA NPs, whereas the use of alginate (Alg) allows efficient Tb entrapment within NPs and its release up to one month. Optimized formulations display good in vitro antimicrobial activity against P. aeruginosa planktonic cells. Furthermore, spray-drying of the NPs with lactose yielded NEM with peculiar but promising flow and aerosolization properties, while preserving the peculiar NP features. Nonetheless, in vivo biodistribution studies showed that PVA-modified Alg/PLGA NPs reached the deep lung, while CS-modified NPs were found in great amounts in the upper airways, lining lung epithelial surfaces. In conclusion, PLGA NP composition appears to play a crucial role in determining not only the technological features of NPs but, once processed in the form of NEM, also their in vitro/in vivo deposition pattern.
Amphiphilic PEG-PCL-PEI triblock copolymers self-assemble into nano-scaled, positively charged, multifunctional carriers, suitable for drug and gene delivery. A set of block copolymers with varying hydrophilic/hydrophobic ratio (systematically altered at the borderline of micelle and particle forming polymers) was synthesized, characterized and assembled into carriers. A detailed structural characterization in the liquid state of these assemblies was carried out: carrier size was determined using dynamic light scattering, cryogenic scanning electron microscopy and atomic force microscopy. Nuclear magnetic resonance analyses elucidated carrier's core-shell structure. ζ-potential and thickness of the hydrophilic outer polymer shell were determined by laser Doppler anemometry. Subsequently the impact of carrier's structure on its features (stability and toxicity) was investigated. Polymers hydrophilic in nature formed small (<40 nm) micelle-like carriers, whilst hydrophobic polymers aggregated to larger particle-like assemblies (>100 nm). Monitoring carrier size as a function of initial polymer concentration clarified different assembly mechanisms. Shell thickness, colloidal stability and toxicity were found to depend on the length of the hydrophilic polymer block. Due to controllable size, charge, stability and toxicity, this class of novel carriers is a promising candidate for prospective co-delivery of drugs and nucleic acids.
The past couple of years have witnessed a tremendous progress in the development of glutathione-responsive nano-vehicles for targeted intracellular drug and gene delivery, as driven by the facts that (i) many therapeutics (e.g. anti-cancer drugs, photosensitizers, and anti-oxidants) and biotherapeutics (e.g. peptide and protein drugs, and siRNA) exert therapeutical effects only inside cells like the cytosol and cell nucleus, and (ii) several intracellular compartments such as cytosol, mitochondria, and cell nucleus contain a high concentration of glutathione (GSH) tripeptides (about 2-10 mM), which is 100 to 1000 times higher than that in the extracellular fluids and circulation (about 2-20 μM). Glutathione has been recognized as an ideal and ubiquitous internal stimulus for rapid destabilization of nano-carriers inside cells to accomplish efficient intracellular drug release. In this paper, we will review recent results on GSH-responsive nano-vehicles in particular micelles, nanoparticles, capsules, polymersomes, nanogels, dendritic and macromolecular drug conjugates, and nano-sized nucleic acid complexes for controlled delivery of anti-cancer drugs (e.g. doxorubicin and paclitaxel), photosensitizers, anti-oxidants, peptides, protein drugs, and nucleic acids (e.g. DNA, siRNA, and antisense oligodeoxynucleotide). The unique disulfide chemistry has enabled novel and versatile designs of multifunctional delivery systems addressing both intracellular and extracellular barriers. We are convinced that GSH-responsive nano-carrier systems have enormous potential in targeted cancer therapy.
Multifunctional and modular block copolymers prepared from biocompatible monomers and linked by a bioreducible disulfide linkage have been prepared using a combination of ring-opening and atom-transfer radical polymerizations (ATRP). The presence of terminal functionality via ATRP allowed cell-targeting folic acid groups to be attached in a controllable manner, while the block copolymer architecture enabled well-defined nanoparticles to be prepared by a water-oil-water double emulsion procedure to encapsulate DNA with high efficiency. Gene delivery assays in a Calu-3 cell line indicated specific folate-receptor-mediated uptake of the nanoparticles, and triggered release of the DNA payload via cleavage of the disulfide link resulted in enhanced transgene expression compared to nonbioreducible analogues. These materials offer a promising and generic means to deliver a wide variety of therapeutic payloads to cells in a selective and tunable way.
Mucus is a viscoelastic and adhesive gel that protects the lung airways, gastrointestinal (GI) tract, vagina, eye and other mucosal surfaces. Most foreign particulates, including conventional particle-based drug delivery systems, are efficiently trapped in human mucus layers by steric obstruction and/or adhesion. Trapped particles are typically removed from the mucosal tissue within seconds to a few hours depending on anatomical location, thereby strongly limiting the duration of sustained drug delivery locally. A number of debilitating diseases could be treated more effectively and with fewer side effects if drugs and genes could be more efficiently delivered to the underlying mucosal tissues in a controlled manner. This review first describes the tenacious mucus barrier properties that have precluded the efficient penetration of therapeutic particles. It then reviews the design and development of new mucus-penetrating particles that may avoid rapid mucus clearance mechanisms, and thereby provide targeted or sustained drug delivery for localized therapies in mucosal tissues.
A series of poly(D,L-lactic-co-glycolic acid) (PLGA)/poly(ethyleneglycol) (PEG) di-block copolymers were synthesized by ring-opening polymerization of D,L-lactide and glycolide with different molecular weights of monomethoxy polyethyleneglycol (mPEG) 750, 2000 and 5000 as an initiator. The bulk properties of these co-polymers were characterized by using 1H NMR spectroscopy, gel permeation chromatography, differential scanning calorimetry (DSC). Electron spectroscopy for chemical analysis (ESCA) results, in which the blend films with the di-block copolymers showed increasing surface oxygen atomic percentage with increasing PEG chain length, indicate that PEG chain segment in the di-block copolymers is surface oriented and enriched onto the surface of the blend films. The extent of protein adsorption onto the surface of these blend films was studied, using iodine radio-labeled human serum albumin, gamma globulin and human growth hormone. The protein adsorption amount was reduced for the blend films prepared with PLGA/PEG 750 and 2000 di-block copolymers, but increased to a great extent for PLGA/PEG 5000 di-block copolymer. This is due to the increased water uptake capacity of the blend film, which absorbed more protein molecules into a swollen polymer matrix in addition to surface adsorption.
The objective of this study was to examine the uptake mechanisms of fluorescent polystyrene microspheres of various diameters and surface chemistry by two human cell lines derived from the respiratory epithelium, A549 and Calu-3.
These results suggest that A549 and Calu-3 cells can internalize microspheres and that size and effective charge played an important role in the uptake process.
The first disulfide linkage-employing drug conjugate that exploits the reversible nature of this unique covalent bond was recently approved for human use. Increasing numbers of drug formulations that incorporate disulfide bonds have been reported, particularly in the next generation macromolecular pharmaceuticals. These are designed to exploit differences in the reduction potential at different locations within and upon cells. The recent characterization of a novel redox enzyme in endosomes and lysosomes adds more excitement to this approach. This review focuses on understanding where and how the disulfide bond in the bioconjugate is reduced upon contact with biological milieu, which affects delivery design and the interpretation of the delivery strategies.
Targeted delivery of cytotoxic agents to tumours is believed to improve both their anti-tumour efficacy and their safety. Antibodies specific for tumour-associated antigens have been used to deliver cytotoxic agents to tumour cells. Calicheamicin is a potent cytotoxic agent that causes double-strand DNA breaks, resulting in cell death. When conjugated to monoclonal antibodies specific for tumour-associated antigens, calicheamicin exerts strong antigen-specific anti-tumour effects against human tumour xenografts in preclinical models. Antibody-targeted chemotherapy with immunoconjugates of calicheamicin, exemplified by gemtuzumab ozogamicin (Mylotarg), is a clinically validated therapeutic strategy for the treatment of human cancer.
We demonstrate quantitative vibrational imaging of specific lipid molecules in single bilayers using laser-scanning coherent anti-Stokes Raman scattering (CARS) microscopy with a lateral resolution of 0.25 mum. A lipid is spectrally separated from other molecules by using deuterated acyl chains that provide a large CARS signal from the symmetric CD(2) stretch vibration around 2100 cm(-1). Our temperature control experiments show that d62-DPPC has similar bilayer phase segregation property as DPPC when mixing with DOPC. By using epi-detection and optimizing excitation and detection conditions, we are able to generate a clear vibrational contrast from d62-DPPC of 10% molar fraction in a single bilayer of DPPC/d62-DPPC mixture. We have developed and experimentally verified an image analysis model that can derive the relative molecular concentration from the difference of the two CARS intensities measured at the peak and dip frequencies of a CARS band. With the above strategies, we have measured the molar density of d62-DPPC in the coexisting domains inside the DOPC/d62-DPPC (1:1) supported bilayers incorporated with 0-40% cholesterol. The observed interesting changes of phospholipid organization upon addition of cholesterol to the bilayer are discussed.
We found that the topology of cleavable polymer from linear to hyperbranched can be tuned simply by varying the polymerization temperature: linear polymers were produced at temperatures <= 40 degrees C, hyperbranched polymers were obtained at elevated temperatures (>= 48 degrees C); the degree of branching (DB) of hyperbranched polymers increased with the increase of temperature. Furthermore, the produced linear and hyperbranched polymers contain stimuli-sensitive disulfide bonds in the backbone that can be easily cleaved into small organic molecules in the presence of DTT.
Nanotechnology has shown tremendous promise in target-specific delivery of drugs and genes in the body. Although passive and active targeted-drug delivery has addressed a number of important issues, additional properties that can be included in nanocarrier systems to enhance the bioavailability of drugs at the disease site, and especially upon cellular internalization, are very important. A nanocarrier system incorporated with stimuli-responsive property (e.g., pH, temperature, or redox potential), for instance, would be amenable to address some of the systemic and intracellular delivery barriers. In this review, we discuss the role of stimuli-responsive nanocarrier systems for drug and gene delivery. The advancement in material science has led to design of a variety of materials, which are used for development of nanocarrier systems that can respond to biological stimuli. Temperature, pH, and hypoxia are examples of "triggers" at the diseased site that could be exploited with stimuli-responsive nanocarriers. With greater understanding of the difference between normal and pathological tissues and cells and parallel developments in material design, there is a highly promising role of stimuli-responsive nanocarriers for drug and gene delivery in the future.
It is generally assumed that polymeric micelles, upon administration into the blood stream, carry drug molecules until they are taken up into cells followed by intracellular release. The current work revisits this conventional wisdom. The study using dual-labeled micelles containing fluorescently labeled copolymers and hydrophobic fluorescent probes entrapped in the polymeric micelle core showed that cellular uptake of hydrophobic probes was much faster than that of labeled copolymers. This result implies that the hydrophobic probes in the core are released from micelles in the extracellular space. Förster resonance energy transfer (FRET) imaging and spectroscopy were used to monitor this process in real time. A FRET pair, DiIC(18(3)) and DiOC(18(3)), was loaded into monomethoxy poly(ethylene glycol)-block-poly(d,l-lactic acid) micelles. By monitoring the FRET efficiency, release of the core-loaded probes to model membranes was demonstrated. During administration of polymeric micelles to tumor cells, a decrease of FRET was observed both on the cell membrane and inside of cells, indicating the release of core-loaded probes to the cell membrane before internalization. The decrease of FRET on the plasma membrane was also observed during administration of paclitaxel-loaded micelles. Taken together, our results suggest a membrane-mediated pathway for cellular uptake of hydrophobic molecules preloaded in polymeric micelles. The plasma membrane provides a temporal residence for micelle-released hydrophobic molecules before their delivery to target intracellular destinations. A putative role of the PEG shell in the molecular transport from micelle to membrane is discussed.
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