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Dendrimer-Drug Conjugates for Tailored Intracellular Drug Release Based on Glutathione Levels
Abstract
N-Acetyl-L-cysteine (NAC) is an antioxidant and anti-inflammatory agent with significant potential in clinical applications including stroke and neuroinflammation. The drug shows high plasma binding upon IV administration, requiring high doses and associated side effects. Through the use of an appropriate delivery vehicle, the stability and efficacy of NAC can be significantly improved. Dendrimers are an emerging class of nanoscale drug delivery vehicles, which enable high drug payloads and intracellular delivery. Poly(amidoamine) (PAMAM) dendrimer-NAC conjugates having cleavable disulfide linkages are designed for intracellular delivery based on glutathione levels. We have successfully synthesized two conjugates with a cationic G4-NH(2) and an anionic G3.5-COOH PAMAM dendrimer with NAC payloads of 16 and 18 per dendrimer, respectively, as confirmed by (1)H NMR and MALDI-TOF analysis. NAC release from the conjugates at intracellular and extracellular glutathione (GSH) concentrations were evaluated by reverse phase HPLC (RP-HPLC) analysis, and approximately 70% of NAC payload was released within one hour at intracellular GSH concentrations (approximately 10 mM), whereas negligible NAC release was observed at extracellular GSH levels (2 microM). FITC-labeled conjugates showed that they enter cells rapidly and localize in the cytoplasm of lipopolysaccharide (LPS)-activated microglial cells (the target cells in vivo). The significantly improved efficacies of dendrimer-NAC conjugates in activated microglial cells was confirmed by measuring the nitrite inhibition in the cell culture medium, which is an indication of the antioxidative property of the drug. Both G4-NH(2) and G3.5-COOH conjugates showed significantly better nitrite inhibition both at 24 and 72 h compared to free NAC, by as much as a factor of 16. The results indicate that PAMAM dendrimer conjugates produce higher local NAC concentration inside the cells, with GSH-sensitive disulfide linker enabling efficient and rapid cellular release of the drug.
... 81 N-Acetylcysteine (NAC) (see Fig. 10A), a glutathione precursor with strong antioxidant, pro-neurogenesis, and anti-inflammatory properties, has acquired considerable potential in clinical applications for stroke, neuroinflammation, and neurodegenerative diseases. 82,83 However, NAC must be administered in higher and repetitive doses. Due to the presence of unbound sulfhydryl groups in NAC, which are capable of spontaneous oxidation and forming disulphide bonds with plasma proteins, NAC has poor bioavailability and blood stability. ...
... Due to the presence of unbound sulfhydryl groups in NAC, which are capable of spontaneous oxidation and forming disulphide bonds with plasma proteins, NAC has poor bioavailability and blood stability. 82 Early pharmacokinetic studies have demonstrated limited oral bioavailability of NAC (in the range of 6% and 10%), which was attributed to low blood concentrations of NAC. 84,85 The administration of large doses can result in cytotoxicity and adverse effects, such as elevated blood pressure. ...
Alzheimer's disease (AD) is a progressive, neurodegenerative condition. There are clear markers for the presence and progression of the disease, including β-amyloid (Aβ) plaques and Tau tangles, with many potential causes debated in the scientific community. Most existing treatments only provide symptomatic solutions. Due to poor aqueous solubility and possibly limited uptake across the blood–brain barrier (BBB), medications targeting the hallmarks of AD are still under study despite enormous efforts. Recently, nanoparticle-based drug delivery systems have demonstrated remarkable promise as precision medicines that may effectively increase bioavailability, permeate the BBB, and improve the targeting ability of a variety of pharmaceuticals. Polymer therapeutics have made tremendous progress in recent years, particularly in cancer treatment. Polymer–drug conjugates (PDCs) typically have a longer half-life, higher stability, and enhanced water solubility. Polymers serve as carriers for the administration of drugs, proteins, targeting moieties, and imaging agents in polymeric and macromolecular prodrugs. Numerous commercially viable PDCs for the treatment of various diseases have already proved their potential. This paper focuses mainly on the rationale for the design, synthesis, and potential use of PDCs as a multi-target treatment for neurodegenerative diseases.
... The solvents and reagents for synthesis were obtained from Merck, TCI Europe, Alfa Aesar, or AK Scientific Inc. The 3 ,5 -Bis-O-(tert-butyldimethylsilyl)thymidine (2) [36] and 3-[(pyridin-2-yl)disulfanyl]propanoic acid (6) [37] were prepared according to the indicated literature methods. Their characterization data were identical to the ones provided in the literature references. ...
... THF. The resulting amine 5 was coupled with 3-(2-tert-butyldisulfanyl)-propanoic acid (7) [51], which was prepared according to Navath et al. [37] (Scheme 1b). As a result, the tert-butyl-protected disulfide functional group in nucleoside 8 was obtained. ...
Oligonucleotide conjugates are versatile scaffolds that can be applied in DNA-based screening platforms and ligand display or as therapeutics. Several different chemical approaches are available for functionalizing oligonucleotides, which are often carried out on the 5′ or 3′ end. Modifying oligonucleotides in the middle of the sequence opens the possibility to ligate the conjugates and create DNA strands bearing multiple different ligands. Our goal was to establish a complete workflow that can be applied for such purposes from monomer synthesis to templated ligation. To achieve this, a monomer is required with an orthogonal functional group that can be incorporated internally into the oligonucleotide sequence. This is followed by conjugation with different molecules and ligation with the help of a complementary template. Here, we show the synthesis and the application of a thiol-modified thymidine nucleoside phosphoramidite to prepare ligatable oligonucleotide conjugates. The conjugations were performed both in solution and on solid phase, resulting in conjugates that can be assembled into multivalent oligonucleotides decorated with tissue-targeting peptides using templated ligation.
... PANAM dendrimers have been used to deliver antioxidants and anti-inflammatory molecules, such as N-acetyl cysteine [201]. In in vitro studies, these NPs attenuated the production of free radical nitric oxide (NO) from the microglia in response to lipopolysaccharide (a very common pro-inflammatory molecule). ...
... Thus, for example, inhibitors of iNOS and nNOS have been shown to reduce cerebral edema and infarct sizes in different animal models. In comparison with the application of free N-acetyl cysteine, the sustained release of N-acetyl cysteine through PANAM dendrimers produces significant reductions in NO production, requiring fewer doses of N-acetyl cysteine to achieve the same effects induced by the administration of free anti-oxidant molecules [201]. These types of strategies based on the use of antioxidants released through biocompatible polymers are not only of interest for stroke but also to treat other brain disorders in which inflammation and oxidative stress influence the course of the disease [202]. ...
Ischemic stroke represents one of the most prevalent pathologies in humans and is a leading cause of death and disability. Anti-thrombolytic therapy with tissue plasminogen activator (t-PA) and surgical thrombectomy are the primary treatments to recanalize occluded vessels and normalize the blood flow in ischemic and peri-ischemic regions. A large majority of stroke patients are refractory to treatment or are not eligible due to the narrow time window of therapeutic efficacy. In recent decades, we have significantly increased our knowledge of the molecular and cellular mechanisms that inexorably lead to progressive damage in infarcted and peri-lesional brain areas. As a result, promising neuroprotective targets have been identified and exploited in several stroke models. However, these considerable advances have been unsuccessful in clinical contexts. This lack of clinical translatability and the emerging use of biomaterials in different biomedical disciplines have contributed to developing a new class of biomaterial-based systems for the better control of drug delivery in cerebral disorders. These systems are based on specific polymer formulations structured in nanoparticles and hydrogels that can be administered through different routes and, in general, bring the concentrations of drugs to therapeutic levels for prolonged times. In this review, we first provide the general context of the molecular and cellular mechanisms impaired by cerebral ischemia, highlighting the role of excitotoxicity, inflammation, oxidative stress, and depolarization waves as the main pathways and targets to promote neuroprotection avoiding neuronal dysfunction. In the second part, we discuss the versatile role played by distinct biomaterials and formats to support the sustained administration of particular compounds to neuroprotect the cerebral tissue at risk of damage.
... Typically, NAC is used between 0.0408-1.306 mg/ml (0.25-8 mM) concentration range for suppression of ROS in the literature [43,67]. Here, the studied dose of free or bound NAC is 0.037-73.2 ...
... μg/ml. So, conjugation of NAC onto nanoparticles increases its ROS scavenging effect probably by improving its uptake as also shown in other reports [43,49,67]. This is interesting since NAC-Ag 2 S lacks free sulfhydryl group, and QDs were stable in lysosomal pH. ...
... N-Acetylcysteine (NAC) is a mucolytic, anti-inflammatory, and hepaprotective agent used as a powerful antioxidant to protect cells [1,2] and to treat diseases such as cancer, neuropsychiatric disorders, and cardiovascular diseases, among others [3,4]. Despite all these benefits, NAC has a low bioavailability (6 to 8%), which limits its therapeutic effects. is happens because, once it enters the bloodstream, it joins the plasmatic proteins and creates disulfide bridges [5]. When supplied intravenously, nearly 30% of NAC is eliminated through, urine and high doses can increase the blood pressure [5,6]. ...
... Despite all these benefits, NAC has a low bioavailability (6 to 8%), which limits its therapeutic effects. is happens because, once it enters the bloodstream, it joins the plasmatic proteins and creates disulfide bridges [5]. When supplied intravenously, nearly 30% of NAC is eliminated through, urine and high doses can increase the blood pressure [5,6]. us, the development of carriers to transport and stabilize NAC inside the body is a major concern when researching for new ways to increase its bioavailability. ...
N-Acetylcysteine (NAC) is a hydrophilic compound with a low bioavailability. It has been used as an effective antioxidant agent. This research seeks to enhance the entrapment of NAC in PLGA nanoparticles for drug delivery systems. The nanoparticles were made using the nanoprecipitation method and changing the following parameters: the solvent/nonsolvent nature, its viscosity, pH, NAC addition to the nonsolvent, the polymer concentration and molecular weight, and NAC concentration in the solvent. The results showed that an increase in the nonsolvent viscosity produces NAC concentration in the solvent, and the nonsolvent rises its entrapment in the nanoparticles. Nanoparticles with 235.5 ± 11.4 nm size with an entrapment efficiency of 0.4 ± 0.04% and a specific load of 3.14 ± 0.33% were obtained. The results suggest that besides efficiently entrapping hydrophobic compounds, the nanoprecipitation method also has a high potential as an alternative entrapment method for hydrophilic compounds as well. However, its use in the pharmaceutical industry, as a proper specific load vehicle, still depends on the improvement of the load capacity.
... The branched structure of dendrimers results in the creation of internal cavities within the nanoparticles, allowing for non-covalent physical entrapment of hydrophobic drugs and their protection from the external environment until reaching their target destination. [147][148][149][150] Dendrimers can also be functionalized with drugs, [151][152][153][154] imaging agents, 153,155,156 and targeting moieties 153,157 via covalent attachment, making them effective drug carriers that can be engineered to release their payload in response to target environment stimuli. Dendrimers can both extend drug bioavailability and facilitate the transport of cargo across the disrupted BBB. ...
Injury and disease in the central nervous system (CNS) can result in a dysregulated inflammatory environment that inhibits the repair of functional tissue. Biomaterials present a promising approach to tackle this complex inhibitory environment and modulate the mechanisms involved in neuroinflammation to halt the progression of secondary injury and promote the repair of functional tissue. In this review, we will cover recent advances in biomaterial strategies, including nanoparticles, hydrogels, implantable scaffolds, and neural probe coatings, that have been used to modulate the innate immune response to injury and disease within the CNS. The stages of inflammation following CNS injury and the main inflammatory contributors involved in common neurodegenerative diseases will be discussed, as understanding the inflammatory response to injury and disease is critical for identifying therapeutic targets and designing effective biomaterial-based treatment strategies. Biomaterials and novel composites will then be discussed with an emphasis on strategies that deliver immunomodulatory agents or utilize cell–material interactions to modulate inflammation and promote functional tissue repair. We will explore the application of these biomaterial-based strategies in the context of nanoparticle- and hydrogel-mediated delivery of small molecule drugs and therapeutic proteins to inflamed nervous tissue, implantation of hydrogels and scaffolds to modulate immune cell behavior and guide axon elongation, and neural probe coatings to mitigate glial scarring and enhance signaling at the tissue–device interface. Finally, we will present a future outlook on the growing role of biomaterial-based strategies for immunomodulation in regenerative medicine and neuroengineering applications in the CNS.
... Although dendrimers can offer enhanced binding of targeting ligands, their capability of drug loading is limited. Dendrimers do not have an internal core that can retain the encapsulation of drugs; therefore, drugs must be conjugated to the surface, complicating the nanoparticle design [77]. To combat this issue, a novel dendron-lipid micelle constructed from generation 3 poly(amidoamine) dendron and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) was developed to encapsulate the drug doxorubicin while maintaining gene delivery properties [69]. ...
Introduction:
Cancer immunotherapies have created a new generation of therapeutics to employ the immune system to attack cancer cells. However, these therapies are typically based on biologics that are non-specific and often exhibit poor tumor penetration and dose-limiting toxicities. Nanocarriers allow the opportunity to overcome these barriers as they have the capabilities to direct immunomodulating drugs to tumor sites via passive and active targeting, decreasing potential adverse effects from non-specific targeting. In addition, nanocarriers can be multifunctionalized to deliver multiple cancer therapeutics in a single drug platform, offering synergistic potential from co-delivery approaches.
Areas covered:
This review focuses on the delivery of cancer therapeutics using emerging nanocarriers to achieve synergistic results via co-delivery of immune-modulating components (i.e. chemotherapeutics, monoclonal antibodies, and genes).
Expert opinion:
Nanocarrier-mediated delivery of combinatorial immunotherapy creates the opportunity to fine-tune drug release while achieving superior tumor targeting and tumor cell death, compared to free drug counterparts. As these nanoplatforms are constantly improved upon, combinatorial immunotherapy will afford the greatest benefit to treat an array of tumor types while inhibiting cancer evasion pathways.
... OP-101 also has been evaluated in phase 1 studies in healthy human volunteers (NCT03500627, NCT04321980). OP-101 is a covalent conjugate of generation-4 hydroxyl terminated poly(amidoamine) dendrimer (~4 nm in size) and N-acetyl cysteine (NAC), enabled by a disulfide linker that readily releases the drug through the action of intracellular glutathione (21,22). Preclinical studies have shown that intravenously delivered hydroxyl polyamidoamine (PAMAM) dendrimers and their conjugates selectively localize in activated microglia/macrophages in the CNS in small and large animal models and deliver drugs to the site of injury producing positive therapeutic outcomes (14,(18)(19)(20). ...
Hyperinflammation triggered by SARS-CoV-2 is a major cause of disease severity, with activated macrophages implicated in this response. OP-101, a hydroxyl-polyamidoamine dendrimer– N -acetylcysteine conjugate that specifically targets activated macrophages, improves outcomes in preclinical models of systemic inflammation and neuroinflammation. In this multicenter, randomized, double-blind, placebo-controlled, adaptive phase 2a trial, we evaluated safety and preliminary efficacy of OP-101 in patients with severe COVID-19. Twenty-four patients classified as having severe COVID-19 with a baseline World Health Organization seven-point ordinal scale of ≥5 were randomized to receive a single intravenous dose of placebo ( n = 7 patients) or OP-101 at 2 ( n = 6), 4 ( n = 6), or 8 mg/kg ( n = 5 patients). All study participants received standard of care, including corticosteroids. OP-101 at 4 mg/kg was better than placebo at decreasing inflammatory markers; OP-101 at 4 and 8 mg/kg was better than placebo at reducing neurological injury markers, (neurofilament light chain and glial fibrillary acidic protein). Risk for the composite outcome of mechanical ventilation or death at 30 and 60 days after treatment was 71% (95% CI: 29%, 96%) for placebo and 18% (95% CI: 4%, 43%; P = 0.021) for the pooled OP-101 treatment arms. At 60 days, 3 of 7 patients given placebo and 14 of 17 OP-101–treated patients were surviving. No drug-related adverse events were reported. These data show that OP-101 was well tolerated and may have potential to treat systemic inflammation and neuronal injury, reducing morbidity and mortality in hospitalized patients with severe COVID-19.
... Further, glial activation has also been assessed using a radiolabeled TSPO ligand. TSPO binding overall was increased from 5 to 180 days post-CA and CA time correlated with hippocampal TSPO binding at all timepoints.25 Previously our group has shown that D-NAC mitigates the microglial response through both in vivo and in vitro studies,26,27,32,46,47 and this work extends our results to a clinically relevant rodent model of global ischemic injury following CA. ...
Cardiac arrest (CA), the sudden cessation of effective cardiac pumping function, is still a major clinical problem with a high rate of early and long‐term mortality. Post‐cardiac arrest syndrome (PCAS) may be related to an early systemic inflammatory response leading to exaggerated and sustained neuroinflammation. Therefore, early intervention with targeted drug delivery to attenuate neuroinflammation may greatly improve therapeutic outcomes. Using a clinically relevant asphyxia CA model, we demonstrate that a single (i.p.) dose of dendrimer‐N‐acetylcysteine conjugate (D‐NAC), can target “activated” microglial cells following CA, leading to an improvement in post‐CA survival rate compared to saline (86% vs. 45%). D‐NAC treatment also significantly improved gross neurological score within 4 h of treatment (p < 0.05) and continued to show improvement at 48 h (p < 0.05). Specifically, there was a substantial impairment in motor responses after CA, which was subsequently improved with D‐NAC treatment (p < 0.05). D‐NAC also mitigated hippocampal cell density loss seen post‐CA in the CA1 and CA3 subregions (p < 0.001). These results demonstrate that early therapeutic intervention even with a single D‐NAC bolus results in a robust sustainable improvement in long‐term survival, short‐term motor deficits, and neurological recovery. Our current work lays the groundwork for a clinically relevant therapeutic approach to treating post‐CA syndrome.
... The authors [41] optimized the ruthenium nanoparticles obtaining mesoporous ruthenium nanoparticles (MRN) increasing the loading ability of antitumor drugs (28.2%) [94]. In addition, since glutathione (GSH) levels in tumor cells are much higher than in normal cells, drug release based on endogenous GSH is considered to be the most efficient strategy and disulfide bonds are the most commonly used part of the GSH trigger system [95][96][97][98]. Standing on these evidences, the authors [41] proposed the use of MRN RBT-loaded NPs covalently bound to both Tf and AS1411 with the addition of the disulfide bonds to obtain a dual-targeted nanomedicine delivery system for drug delivery to gliomas, which they called RBT@MRN-SSTf/Apt [41]. ...
Glioblastoma (GBM) is the most aggressive, infiltrative, and lethal brain tumor in humans. Despite the extensive advancement in the knowledge about tumor progression and treatment over the last few years, the prognosis of GBM is still very poor due to the difficulty of targeting drugs or anticancer molecules to GBM cells. The major challenge in improving GBM treatment implicates the development of a targeted drug delivery system, capable of crossing the blood-brain barrier (BBB) and specifically targeting GBM cells. Aptamers possess many characteristics that make them ideal novel therapeutic agents for the treatment of GBM. They are short single-stranded nucleic acids (RNA or ssDNA) able to bind to a molecular target with high affinity and specificity. Several GBM-targeting aptamers have been developed for imaging, tumor cell isolation from biopsies, and drug/anticancer molecule delivery to the tumor cells. Due to their properties (low immunogenicity, long stability, and toxicity), a large number of aptamers have been selected against GBM biomarkers and tested in GBM cell lines, while only a few of them have also been tested in in vivo models of GBM. Herein, we specifically focus on aptamers tested in GBM in vivo models that can be considered as new diagnostic and/or therapeutic tools for GBM patients' treatment.
... Many drug conjugated dendrimers and nanodevices were developed against C. trachomatis [63,64]. The same group was consistently working on the design and development of novel dendrimers; such as dendrimers in loading high amount of drugs [65], targeted delivery of drugs (ocular delivery) [66][67][68][69], as sensor for cytokines and other biomarkers [70,71], anti-inflammatory including but not limited to neuroinflammation [72][73][74], lung inflammation [75], brain inflammation [76], and anti-oxidant [77], anti-cancer [78], antimicrobial activities [79], and many other outcomes [80]. ...
... Different neuroprotective molecules with scavenging and anti-oxidant potential have been delivered through different biomaterial scaffolds. This situation applies for example to N-acetyl cysteine delivered from poly (amidoamine) dendrimers (Navath et al., 2008). In this case, N-acetyl cysteine attenuated the production of free radical nitric oxide from inflammatory microglia. ...
The neurological devastation of neurodegenerative and cerebrovascular diseases reinforces our perseverance to find advanced treatments to deal with these fatal pathologies. High-performance preclinical results have failed at clinical level, as it has been the case for a wide variety of neuroprotective agents and cell-based therapies employed to treat high prevalent brain pathologies such as stroke, Alzheimer's and Parkinson's diseases. An unquestionable reality is the current absence of effective therapies to neuroprotect the brain, to arrest neurodegeneration and rewire the impaired brain circuits. Part of the problem might arise from the lack of adequate in vitro and in vivo models and that most of the underlying pathophysiological mechanisms are not yet clarified. Another contributing factor is the lack of efficient systems to sustain drug release at therapeutic concentrations and enhance the survival and function of grafted cells in transplantation procedures. For medical applications the use of biomaterials of different compositions and formats has experienced a boom in the last decades. Although the greater complexity of central nervous system has probably conditioned their extensive use with respect to other organs, the number of biomaterials-based applications to treat the injured brain or in the process of being damaged has grown exponentially. Hydrogel-based biomaterials have constituted a turning point in the treatment of cerebral disorders using a new form of advanced therapy. Hydrogels show mechanical properties in the range of cerebral tissue resulting very suitable for local implantation of drugs and cells. It is also possible to fabricate three-dimensional hydrogel constructs with adaptable mesh size to facilitate axonal guidance and elongation. Along this article, we review the current trends in this area highlighting the positive impact of hydrogel-based biomaterials over the exhaustive control of drug delivery, cell engraftment and axonal reinnervation in brain pathologies.
... [20] (网络版彩图) Figure 1 The CAPIR cascade for a nanomedicine to deliver a free drug into tumor cells [20] (color online). [28,29] 、谷胱甘肽(GSH) [12,30] 、活性氧(ROS) [ ...
... Another NP model that is used as a drug carrier is dendritic NPs, also called dendrimers, included in the polymeric NPs group. Drug-dendrimer combinations are projected to transport therapeutic agents to specific tissues to reduce systemic effects and increase effectiveness in the target locations (Navath et al., 2008). Using an ex vivo perfused human placenta model, one study investigated the potential of using dendrimer-drug combinations as delivery vectors to selectively treat the mother, without affecting the fetus, and described the transplacental transportation distribution of the poly (amidoamine) (PAMAM)-dendrimer marked with fluorescence. ...
During pregnancy, the placenta regulates the transfer of oxygen, nutrients, and residual products between the maternal and fetal bloodstreams and is a key determinant of fetal exposure to xenobiotics from the mother. To study the disposition of substances through the placenta, various experimental models are used, especially the perfused placenta, placental villi explants, and cell lineages models. In this context, nanotechnology, an area of study that is on the rise, enables the creation of particles on nanometric scales capable of releasing drugs aimed at specific tissues. An important reason for furthering the studies on transplacental transfer is to explore the potential of nanoparticles (NPs), in new delivery strategies for drugs that are specifically aimed at the mother, the placenta, or the fetus and that involve less toxicity. Due to the fact that the placental barrier is essential for the interaction between the maternal and fetal organisms, as well as the possibility of NPs being used in the treatment of various pathologies, the aim of this review is to present the main experimental models used in studying the maternal‐fetal interaction and the action of NPs in the placental environment. This article is protected by copyright. All rights reserved.
... The application of N-hydroxysuccinimide (NHS) 57 or members of the carbodiimide family, which includes N,N 0 -diisopropyl carbodiimide (DIC), 58 60 as coupling agents represents the most popular strategies employed to decorate dendrimers. Furthermore, the exploration of bioorthogonal groups for the conjugation of bioactive molecules may allow the development of new dendrimeric structures, while the application of stimuli-responsive linkers (i.e., hydrazone 61 and disulfide bonds) 62 supports the controlled release of bioactive molecules. ...
Well-defined synthetic branched nanostructures form an emerging subclass of macromolecular structures, whose 3D structure and multivalency offer unique opportunities for fine-tuning their internalization and cellular targeting. In particular, dendrimers possess a well-defined 3D-globular backbone with highly versatile functional surface groups and exhibit a range of chemical and biological properties. Branched polymers present unique opportunities for the targeted delivery of diverse bioactive molecules (including targeting ligands, imaging probes, and drugs) via conjugation to multiple sites within the structure. The inherent versatility and multifunctionality of these architectures make them potentially useful for the modulation of multiple immune-related pathways for the treatment of a wide range of disease and disorders, including cancer and human immunodeficiency virus infection. Herein, we describe the key components of the immune system whose targeting can help to overcome immune-related disorders and discuss branched polymers (including dendrimers) as promising delivery systems with unique immunomodulatory properties against cancer and infectious diseases.
... In such cases, drug release is controlled by the careful selection of the cleavable linker, which connects the drug with the rest of the DDS. This prodrug strategy [20] i. e., covalent attachment of a drug with the carrier is broadly utilized to conjugate a drug molecule with peptides, [21] polymers, [22] dendrimers [23] and nanoparticles. [24] Most recently peptide based drug amphiphiles were developed which can self-assemble to form hydrogels [25] as well as various nanostructures for the delivery of antitumor agents. ...
Widely used chemotherapeutic agent 5‐fluorouracil (5‐Fu) exhibits adverse effects by damaging the normal cells. Controlled delivery and release of such antitumor drugs is essential for better treatment. Herein, a low molecular weight peptide‐drug conjugate for the photo‐controlled delivery of 5‐Fu is demonstrated. 5‐Fu is covalently attached with a short peptide through a photo‐cleavable linker. With a careful choice of the peptide sequence, the peptide‐drug conjugate was able to form a hydrogel within a narrow pH window (pH 6.0 ‐ 8.0). MTT assay of the peptide‐drug conjugate in HeLa cells inferred almost no cytotoxicity up to a high concentration of 110 μg/mL. The gelator prodrug releases 5‐Fu in a controlled, does‐dependent manner under irradiation. The characterizations and controlled release of the drug were investigated using various analytical techniques including FTIR, HPLC and UV‐visible spectroscopy whereas, morphology of the gel was studied by FESEM. The gelation behaviour and rheology of the conjugate was also studied in details. Such covalently peptide‐conjugated 5‐Fu gel could be useful for effective delivery of antitumor agent.
... It was established that the DNA transfection performed by dendrimers is several times more efficient than those by other commercial cationic nanoparticles (Peng et al. 2010;Wang et al. 2011). At the same time, the cell enzymatic systems are destroying the nanoparticles (Navath et al. 2008;Jin et al. 2014). ...
The commercial solution of fourth generation (G4) of poly-amidoamine (PAMAM) dendrimers contains methanol, which is toxic for human body. Our differential scanning calorimetry (DSC) study of dendrimers confirmed the existence of this threat. The recommendation is done on how to prepare dendrimer solutions for practical and safely use in gene delivery. DSC have been also used to study the thermodynamic properties of DNA/dendrimer complexes (dendriplexes). We showed that up to DNA/dendrimer ratio 43 ± 3 (w/w) the solution was homogeneous, but stable aggregates were formed at higher PAMAM content. DSC experiments performed with homogeneous solution of dendriplexes revealed existence of the pH-dependent melting curves that contain several endothermic peaks associated with melting of GC-rich regions.
... The conjugates showed about 70% of NAC payload within 1 h in response to intracellular GSH concentrations, however negligible NAC release was seen at extracellular GSH levels. The G4-NH 2 and G3.5-COOH conjugates demonstrated higher nitrite inhibition at 24 h and 72 h in comparison to free NAC (Navath et al., 2008). ...
Dendrimers are novel polymeric nanoarchitectures characterized by hyper-branched 3D-structure having multiple functional groups on the surface that increases their functionality and make them versatile and biocompatible. Their unique properties like nanoscale uniform size, high degree of branching, polyvalency, water solubility, available internal cavities and convenient synthesis approaches make them promising agent for biological and drug delivery applications. Dendrimers have received an enormous attention from researchers among various nanomaterials. Dendrimers can be used as a carrier for diverse therapeutic agents. They can be used for reducing drug toxicities and enhancement of their efficacies. The present review provide a comprehensive outline of synthesis of dendrimers, interaction of dendrimer with guest molecules, properties, characterization and their potential applications in pharmaceutical and biomedical field.
... Degradation may be selective or non-selective, for example, the hydrolysis of ester linkages is nonspecific and occurs under all physiological conditions, while the reduction of disulfide bonds in the presence of the biological reducing agent L-glutathione (GSH) occurs primarily in intracellular environments as a consequence of the 100-fold increase of GSH in intracellular environments compared to extracellular environments [25,26]. Indeed, the difference in intracellular and extracellular GSH concentration has been elegantly exploited for the disassembly of polymeric constructs through the cleavage of disulfide crosslinks, which has been coupled to the release of a range of payloads [27,28]. However, in all instances of particle disassembly through degradation or breakage of crosslinks, consideration must be given to the resulting degradation products, which may differ significantly in chemical structure, molar mass, solubility, and therefore toxicity and retention, compared to the original polymer. ...
The physical properties of cyclic and linear polymers are markedly different; however, there are few examples which exploit these differences in clinical applications. In this study, we demonstrate that self-assemblies comprised of cyclic-linear graft copolymers are significantly more stable than the equivalent linear-linear graft copolymer assemblies. This difference in stability can be exploited to allow for triggered disassembly by cleavage of just a single bond within the cyclic polymer backbone, via disulfide reduction, in the presence of intracellular levels of l-glutathione. This topological effect was exploited to demonstrate the first example of topology-controlled particle disassembly for the controlled release of an anti-cancer drug in vitro. This approach represents a markedly different strategy for controlled release from polymer nanoparticles and highlights for the first time that a change in polymer topology can be used as a trigger in the design of delivery vehicles. We propose such constructs, which demonstrate disassembly behavior upon a change in polymer topology, could find application in the targeted delivery of therapeutic agents.
... Linkages designed to be selectively cleaved by triggers that are specific to the tumor microenvironment such as acidic pH and enzymes can be more useful for targeted drug delivery (Fig. 2). Disulfide bond was effective for intracellular delivery of N-acetyl cysteine (NAC) (Navath et al. 2008;Kurtoglu et al. 2009;Nance et al. 2017). When NAC was conjugated to PAMAM dendrimer via disulfide bond that is cleavable by glutathione (GSH) within the cytoplasm, the conjugate showed effective release of NAC at intracellular GSH level with superior efficacy compared to free NAC. ...
Dendrimers offer well-defined nanoarchitectures with spherical shape, high degree of molecular uniformity, and multiple surface functionalities. Such unique structural properties of dendrimers have created many applications for drug and gene delivery, nanomedicine, diagnostics, and biomedical engineering. Dendrimers are not only capable of delivering drugs or diagnostic agents to desired sites by encapsulating or conjugating them to the periphery, but also have therapeutic efficacy in their own. When compared to traditional polymers for drug delivery, dendrimers have distinct advantages, such as high drug-loading capacity at the surface terminal for conjugation or interior space for encapsulation, size control with well-defined numbers of peripheries, and multivalency for conjugation to drugs, targeting moieties, molecular sensors, and biopolymers. This review focuses on recent applications of dendrimers for the development of dendrimer-based nanomedicines for cancer, inflammation, and viral infection. Although dendrimer-based nanomedicines still face some challenges including scale-up production and well-characterization, several dendrimer-based drug candidates are expected to enter clinical development phase in the near future.
... At physiological conditions (PBS 7.4, 37°C; data not shown) and in plasma (37°C), the D-NAC conjugate was stable and did not release NAC over a 24-h period (Additional file 3). However, at intracellular GSH concentrations (250 μM), the conjugate released the drug readily within 3.5 h (Additional file 3), indicating that the use of a disulfide linker enables rapid release of NAC from the conjugate only when it is exposed to an intracellular GSH-rich environment [46,47]. Interestingly, D-NAC was stable in human plasma (with 10 μM GSH) at 37°C over a period of 24 h, releasing less than 3% of NAC (Additional file 3). ...
Background
Rett syndrome (RTT) is a pervasive developmental disorder that is progressive and has no effective cure. Immune dysregulation, oxidative stress, and excess glutamate in the brain mediated by glial dysfunction have been implicated in the pathogenesis and worsening of symptoms of RTT. In this study, we investigated a new nanotherapeutic approach to target glia for attenuation of brain inflammation/injury both in vitro and in vivo using a Mecp2-null mouse model of Rett syndrome. Methods
To determine whether inflammation and immune dysregulation were potential targets for dendrimer-based therapeutics in RTT, we assessed the immune response of primary glial cells from Mecp2-null and wild-type (WT) mice to LPS. Using dendrimers that intrinsically target activated microglia and astrocytes, we studied N-acetyl cysteine (NAC) and dendrimer-conjugated N-acetyl cysteine (D-NAC) effects on inflammatory cytokines by PCR and multiplex assay in WT vs Mecp2-null glia. Since the cysteine-glutamate antiporter (Xc−) is upregulated in Mecp2-null glia when compared to WT, the role of Xc− in the uptake of NAC and l-cysteine into the cell was compared to that of D-NAC using BV2 cells in vitro. We then assessed the ability of D-NAC given systemically twice weekly to Mecp2-null mice to improve behavioral phenotype and lifespan. ResultsWe demonstrated that the mixed glia derived from Mecp2-null mice have an exaggerated inflammatory and oxidative stress response to LPS stimulation when compared to WT glia. Expression of Xc− was significantly upregulated in the Mecp2-null glia when compared to WT and was further increased in the presence of LPS stimulation. Unlike NAC, D-NAC bypasses the Xc− for cell uptake, increasing intracellular GSH levels while preventing extracellular glutamate release and excitotoxicity. Systemically administered dendrimers were localized in microglia in Mecp2-null mice, but not in age-matched WT littermates. Treatment with D-NAC significantly improved behavioral outcomes in Mecp2-null mice, but not survival. Conclusions
These results suggest that delivery of drugs using dendrimer nanodevices offers a potential strategy for targeting glia and modulating oxidative stress and immune responses in RTT.
... A variety of design approaches via the structural modification of PAMAM dendrimers and their functionalization have been used to optimize the drug release and activity and provide higher intracellular drug concentrations [75,76]. Application of trastuzumab-grafted dendrimers has resulted in the improved delivery of docetaxel and higher antiproliferation activity towards HER2-positive breast cancer cells [77]. ...
The limited efficiency of the conventional treatment options against a variety of disorders have created demands towards the development of more efficient theranostic strategies. Advances in nanomedicine have provided promising perspectives for the treatment of hard-to-treat diseases. Application of the high-resolution imaging, multifunctional nanoparticles, and a wide variety of nanodevices for early diagnosis of disease, promoting tissue regeneration, and targeted delivery of therapeutic agents, may result in minimal side effects and improved treatment outcome. Meanwhile, a number of nanotherapeutics have shown poor efficiency that might be due to a variety of factors including the inappropriate design protocols or nonspecific drug release. Selective cell targeting and controlled drug release are the key elements which should be considered in designing the nanocarriers for targeted delivery of therapeutic agents. Linkers, the essential components incorporated in the building blocks of drug delivery systems, provide the stability, high payloads, targeted drug delivery, and controlled release, hence, they might be critically involved in the nanotherapeutic activity. In the present review, the significance of linkers in the development of effective drug delivery systems has been highlighted.
Ischemic stroke (IS) affects 15 million people globally which leads to death or disability. Currently, Tissue plasminogen activator (tPA) is the only medication approved by the US Food and Drug Administration (FDA) for treating IS. This treatment eliminates the brain’s blood shortage and the reperfusion-related adverse effects that cause significant tissue damage. Therefore, novel treatment approaches are urgently needed to preserve the integrity of the blood-brain barrier (BBB) and salvageable brain tissue. Nanomedicine opens a new door for emerging strategies that can be promising therapeutic approaches for IS. This chapter first discusses the pathophysiology and different events of IS, then available therapeutic interventions for IS followed by different nanocarriers available. This chapter also covers viral vectors and extracellular vesicles for IS. Also, it focuses on the intranasal administration of nanomedicines, which might cross the BBB and finally discusses toxicity related to nanocarriers for IS. This chapter is completely focused on using nanomedicines for the use of IS.
Drug delivery systems (DDSs) triggered by local microenvironment represents the state-of-art of nanomedicine design, where the triggering hallmarks at intracellular and subcellular levels could be employed to exquisitely recognize the diseased sites, reduce side effects, and expand the therapeutic window by precisely tailoring the drug-release kinetics. Though with impressive progress, the DDS design functioning at microcosmic levels is fully challenging and underexploited. Here, we provide an overview describing the recent advances on stimuli-responsive DDSs triggered by intracellular or subcellular microenvironments. Instead of focusing on the targeting strategies as listed in previous reviews, we herein mainly highlight the concept, design, preparation and applications of intracellular models. Hopefully, this review could give useful hints in developing nanoplatforms proceeding at a cellular level.
In this study, we focus on investigating the therapeutic effects of camptothesome on treating metastatic triple-negative breast cancer (TNBC). We elucidate that camptothesome elicited stronger immunogenic cell death (ICD) compared to free camptothecin (CPT) and Onivyde in 4T1 TNBC cells. In addition, camptothesome is mainly internalized by the 4T1 and MDA-MB-231 cells through clathrin-mediated endocytosis based on the results of flow cytometry. Through real-time Lago optical imaging, camptothesome shows excellent tumor-targeting efficiency in orthotopic TNBC tumors. We demonstrate that camptothesome can upregulate programmed death-ligand 1 (PD-L1) in 4T1 tumors in an interferon gamma (IFN-γ)-dependent manner. Furthermore, the anti-TNBC efficacy studies reveal that camptothesome is superior to Onivyde and markedly potentiates PD-L1 immune checkpoint blockade therapy with complete lung metastasis remission in an orthotopic 4T1-Luc2 tumor model. This combination therapy eliciting robust cytotoxic T lymphocytes (CTL) response via boosting tumor-infiltrating cluster of differentiation 8 (CD8), calreticulin (CRT), high mobility group box 1 protein (HMGB-1), low-density lipoprotein receptor-related protein 1 (LRP1), IFN-γ, and granzyme B. Our work corroborates the promise of camptothesome in favorably modulating tumor immune microenvironment via inducing ICD to fortify the PD-L1 checkpoint blockade therapy for improved treatment of intractable TNBC.
Dendritic polymers are the macromolecular carriers having tree-like three-dimensional structures, which are synthesized following iterative sequence of monomeric conjugation leading to hyperbranched arrangement. The characteristic properties of dendritic macromolecules include monodispersity, multiple surface functionalities, nanometric size, etc., which facilitated these polymers as one of the prominent family of drug delivery carriers or cargos. Along with the traditional synthetic pathways like convergent and divergent, some of the newly developed methods like double exponential growth, hypercores, and branched monomer growth have demonstrated significant productivity in recent years. Dendritic carriers or dendrimers can be categorized into different types on the basis of functionality, biodegradability, and responses toward various stimuli. Some of the well-known classes of dendrimers include poly(amidoamine) (PAMAM), poly(propyleneimine) (PPI), polylysine, poly(ester amide), etc. These drug carriers offer several advantages such as solubility enhancement of hydrophobic drugs, targeted delivery through surface engineering, etc. However the cationic charge associated with dendritic polymers usually poses challenge of in vitro as well as in vivo toxicity. Therapeutic moieties may be delivered to the targeted site through dendritic polymer macromolecular carriers using certain basic approaches, namely encapsulation, conjugation, and electrostatic interactions. Cytotoxicity, hematological toxicity, immunogenicity, and in vivo toxicity put questions over their effectiveness and therefore sometimes these are considered as the dark wave of these carriers. Dendrimers have been utilized successfully to deliver anticancer agents, antimicrobials, gene, etc., via excellent host-guest chemistry. This chapter is focused on the concepts, design, and therapeutic applications of dendritic polymer macromolecular carriers. It includes discussion of key basic properties, synthesis of the concerned macromolecules, their types, associated toxicity, and possible drug delivery approaches. Last part of the chapter is dedicated to the applications of dendritic polymers in the delivery of therapeutic agents for the effective management of various diseases.
Chemotherapy is severely limited by continuously decreased therapeutic efficacy and uncontrolled side effects on normal tissue, which can be improved by constructing a nanoparticle-based drug delivery system (DDS). Nevertheless, no studies have reported on DDS-based on carbon-nanodots (CNDs), combining subcellular organelle-targeted imaging/drug delivery, high drug loading content, and glutathione (GSH)-sensitive drug release into one system. Herein, the as-fabricated CNDs can be covalently conjugated with a mitochondria-targeting ligand (triphenylphosphine, TPP), a smart GSH-responsive disulfide linker (S-S), and the anticancer drug (camptothecin, CPT) to initially prepare a theranostic nano-DDS (TPP-CNDs-S-CPT) with the drug loading efficiency of 64.6 wt %. Owing to excellent water dispersibility, superior fluorescence properties, satisfactory cell permeability, and favorable biocompatibility, TPP-CNDs-S-CPT was successfully used for intracellular mitochondrial-targeted imaging in vitro. High intracellular GSH concentrations in tumor cells caused the cleavage of S-S, resulting in concomitant activation and release of CPT, as well as significant fluorescence enhancement. In vivo, TPP-CNDs-S-CPT exhibited lower biological toxicity and even higher tumor-activatable performance than free CPT, as well as specific cancer therapy with few side effects. The mitochondria-targeted ability and the precise drug-release in tumor make TPP-CNDs-S-CPT a hopeful chemotherapy prodrug, providing significant theoretical basis and data support for in-depth understanding and exploration of chemotherapeutic DDS-based on CNDs.
The development of bioactive components as a delivery system with the use of advanced nanoscience is opening new therapeutic avenues for the management of various diseases. Among recent novel applications, plant phytopharmaceuticals and nutraceuticals are the fastest growing areas of nanotechnology-based research for effective public healthcare. Bioactive compounds, either encapsulated or in entrapped form within novel drug delivery systems are reported as a booster treatment for the various chronic infections and life-threatening diseases, including cancer, cardiovascular disorders, hypertension, diabetes, asthma, malaria, microbial infections, immune disorders, and gastrointestinal disorders. Recently, considerable progress surged in understanding the factors associated with these diseases. A variety of nanoscience-based formulations such as polymeric matrix nanoparticles, aerosol inhalers/nebulizers nanoemulsion, and vesicular carrier systems including liposome, phytosome, transfersome, herbosome, ethosome, niosome, have proven valuable in the delivery of bioactive materials. Moreover, the scientific community had reported that the herbs and herbal bioactive compounds have notable recompense compared to the conventional method of delivering phytopharmaceuticals and plant extracts, with enhanced solubility, bioavailability, stability, tissue distribution, abridged toxicity, improved pharmacological efficacy, and protection from physicochemical degradation. The current chapter focuses on the carrier-based delivery of bioactive as a booster with advanced using nanoscience, such as nanoemulsion and vesicular drug delivery systems. In addition, the chapter also elaborates patented technologies along with potential bioactive products available in the market.
Dendrimers are very small-sized macromolecules with a well-defined and hyperbranched structure which offers high variety of surface functionality. They are polyvalent and this feature provides different kinds of interactions with other components depending on the chemical nature of terminal groups. Nowadays different kinds of dendrimers with various surface-ending groups and different surface charges (negative, positive, and neutral) are established. Dendrimers with high density of surface active groups can be easily functionalized by different moieties including therapeutic agents, targeting ligand, solubilizing agents, imaging and diagnostic units through different strategies. Chemical modification of dendrimer's surface is an easy way to engineer and control their biological interactions. The well-defined molecular structure and their spherical construction provide an attractive architecture for dendrimers and they can be used as multifunctional vector for drug delivery applications.
Ischemic stroke is still a serious threat to human life and health, but there are few therapeutic options available to treat stroke because of limited blood-brain penetration. The development of nanotechnology may overcome some of the problems related to traditional drug development. In this review, we focus on the potential applications of nanotechnology in stroke. First, we will discuss the main molecular pathological mechanisms of ischemic stroke to develop a targeted strategy. Second, considering the important role of the blood-brain barrier in stroke treatment, we also delve mechanisms by which the blood-brain barrier protects the brain, and the reasons why the therapeutics must pass through the blood-brain barrier to achieve efficacy. Lastly, we provide a comprehensive review related to the application of nanomaterials to treat stroke, including liposomes, polymers, metal nanoparticles, carbon nanotubes, graphene, black phosphorus, hydrogels and dendrimers. To conclude, we will summarize the challenges and future prospects of nanomedicine-based stroke treatments.
Smart materials, which can change properties when an external stimulus is applied, can be used for the targeted drug delivery of an active molecule to a specific site in the correct dosage. Different materials such as liposomes, polymeric systems, nanomaterials and hydrogels can respond to different stimuli such as pH, temperature and light and these are all attractive for controlled release applications.
With so many papers available on smart and stimuli-responsive materials for drug delivery applications it's hard to know where to start reading about this exciting topic. This two volume set brings together the recent findings in the area and provides a critical analysis of the different materials available and how they can be applied to advanced drug delivery systems.
With contributions from leading experts in the field, including a foreword from distinguished scientist Nicholas Peppas, The University of Texas at Austin, USA, the book will provide both an introduction to the key areas for graduate students and new researchers in the stimuli-responsive field as well as serving as a reference for those already working on fundamental materials research or drug delivery applications.
Recently, nanotherapeutics has revolutionized the major impact on healthcare strategies and health facilities. Nanotherapeutics includes design, development and application of therapeutic agents having nano-size (1-100 nm). Due to implications in gene therapy and drug delivery, nanotherapeutics has received much interest in the current scenario. The major area where research is going on and giving maximum benefits from nano-based delivery system includes cancer diagnosis and targeting. The nanotherapeutics are designed in such a way that they will overcome the major drawbacks of conventional therapy and have multi-functionalities so that it can be targeted to cancer site. Nanotherapeutics have increased the permeability and retention of anticancer agents thereby targeting them to the tumor site. Nanotherapeutics has increased the effectiveness of anticancer agents with less or no toxicity. Likewise, diagnostic imaging is also possible with fluorescent nanoparticles based nanotherapeutics and has major potential applications in recent upcoming years with newer fluorescent nanomaterials for specific cancer targeting. Background- NTs provided the possibility of delivering drugs to specific cells using nanoparticles. The overall drug consumption and side-effects may be lowered significantly by depositing the active agent in the morbid region only and in no higher dose than needed. Aim- Attempt have been made to define the nanotherapeutics and to compile the latest developments in the field. Because the topic is of high importance to the general human wellbeing and covers recapitulation of cancer nanotherapeutics and summarizes and restates the main points of nanotherapeutics in the treatment of cancer disease.
The past three decades have witnessed an exponential increase in the structural diversity and applications of dendrimers, spanning across drug delivery and diagnostics, protein, and enzyme mimicry, solubility enhancement, coatings, light harvesting, and catalysis. The dendrimer community has recently focused on internally functionalized dendrimers (IFDs) owing to their advanced design and functionality. The synthesis of IFDs relies on advanced orthogonal chemistries and/or (de)protection schemes, as well as careful purification to minimize polydispersity of composition and molecular weight. The studies published on IFDs, however, lay scattered across the chemical literature, and a comprehensive presentation of structural rationale, synthetic procedures, and technologically relevant applications is missing. To address this need, this review presents a comprehensive collection and discussion of all available studies on IFDs, detailing their methods of synthesis and their structure–function correlations. The wide variety of internal functionalities, including hydroxyl, amine, carboxylic acid, allyl, alkyne, and imidazole groups, enables myriad applications in biochemistry, chemical and biomedical engineering, and material science. Particular focus is given to IFDs that are amenable to modular synthetic strategies, which promote higher synthetic yield and scalability, and therefore possess stronger translational and commercial potential. As such, this review guides research groups pursuing the difficult task of IFD rational design and synthesis providing them a concise roadmap to their mission.
A charged synthetic peptide based non-cytotoxic hydrogelator was employed in encapsulation, storage and sustainable release of different kinds of drugs namely, ciprofloxacin (CP), an antibiotic and 5 fluorouracil (5-FU), an anticancer drug and proteins like Lysozyme and BSA. Hydrogelation of the peptide and its coassembly with the drug molecules was studied to mechanistic details. All the different cargos were capable of sustained and efficient release from the delivery platform. The drugs were found to retain their activity post release while the proteins showed complete retention of their secondary structure. While about 80% CP was released at physiological pH over a period of three days, 5-FU was better released (73%) at an acidic pH (5.5) in comparison to the physiological pH (68%). Lysozyme was better released (82%) than BSA (43%) owing to the smaller size of the former and negative charge on the later. Such biocompatible multi-cargo releasing platforms from simple economically viable biomaterials, capable of sustained and tissue specific release of cargo are extremely promising in topical delivery of therapeutics.
In this study, polyhedral oligomeric silsesquioxane-N-acetylcysteine (POSS-NAC) conjugate as a potential antioxidant molecule was synthesized from N-acetylcysteine (NAC) and amino-functional POSS structure by click chemistry. The chemical structures and thermal properties of the synthesised POSS-NAC conjugate was characterized by spectroscopic and thermal analysis techniques. The antioxidant capacity of the POSS-NAC conjugate was also determined by the 2,2’-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activity and reducing power methods. According to the reducing power method, POSS-NAC structure has lower reducing activity than standard ascorbic acid and trolox (p<0.001). It was found from the ABTS radical scavenging activity results that the synthesized POSS-NAC conjugate had a significantly higher radical scavenging effect than the standards (p <0.001). Biocompatibility properties of the POSS-NAC structure were detected in vitro cell culture system with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test on L-929 mouse fibroblast cells. The synthesized POSS-NAC conjugate exhibits high antioxidant activity and good biocompatibility.
In the past decade, a variety of cysteine‐based multifunctional biomaterials has been successfully developed for drug delivery and proven invaluable in campaigns against human disease. Despite several significant achievements, a systematic investigation of the structure–property relationships in the field of drug delivery has not been performed. In this review, a variety of up‐to‐date literature results are discussed and compiled in which cysteine and its derivatives have been used in the preparation of payload delivery systems for the treatment of human disease. Particular emphasis is placed on the synthesis methods, especially the structure–property relationship for drug delivery and disease therapy. Additionally, this review focuses on recent innovations in redox‐responsive nanocarriers based on cysteine and its derivatives for in vivo drug delivery to achieve a better therapeutic effect. In summary, nanocarriers based on cysteine and its derivatives have the potential to enhance the efficiency of human disease therapy without increasing toxicity.
Journal of Nanotechnology. SPECIAL ISSUES OPEN ACCESS . Nanoparticles for Environment, Engineering, and Nanomedicine
In this work, the first synthesis of poly(amidoamine) (PAMAM) dendrimers whose branches are hybridized with peptide segments (DendriPeps) is reported. The intercalation of amino acids within the branches of PAMAMs provides supplementary internal functionalities to the coronal groups. Four DendriPep prototypes are synthesized with lysine or glutamic acid as “guest” amino acids, displaying, respectively, a primary amine or a carboxyl group, on generation (G)2 and G3 PAMAMs as host scaffolds. The precise control over the number, type, and topological placement of functional groups expands the functional behavior of DendriPeps beyond current PAMAM dendrimers toward new frontiers or colloids, drug delivery vectors, and catalysis. The synthesis of dendrimers displaying internal functional groups through the incorporation of peptide segments in a poly(amidoamine) backbone is reported. The synthetic strategy is modular and can be tailored to synthesize dendrimers with a backbone that contains any functional group. Molecular dynamics simulations indicate that this architecture will be suitable for the covalent conjugation of encapsulated “guest” molecules.
Dendritic polyglycerol‐co‐polycaprolactone (PG‐co‐PCL)‐derived block copolymers are synthesized and explored as nanoscale drug delivery platforms for a chemotherapeutic agent, gemcitabine (GEM), which is the cornerstone of therapy for pancreatic ductal adenocarcinoma (PDAC). Current treatment strategies with GEM result in suboptimal therapeutic outcome owing to microenvironmental resistance and rapid metabolic degradation of GEM. To address these challenges, physicochemical and cell‐biological properties of both covalently conjugated and non‐covalently stabilized variants of GEM‐containing PG‐co‐PCL architectures have been evaluated. Self‐assembly behavior, drug loading and release capacity, cytotoxicity, and cellular uptake properties of these constructs in monolayer and in spheroid cultures of PDAC cells are investigated. To realize the covalently conjugated carrier systems, GEM, in conjunction with a tertiary amine, is attached to the polycarbonate block grafted from the PG‐co‐PCL core. It is observed that pH‐dependent ionization properties of these amine side‐chains direct the formation of self‐assembly of block copolymers in the form of nanoparticles. For non‐covalent encapsulation, a facile “solvent‐shifting” technique is adopted. Fabrication techniques are found to control colloidal and cellular properties of GEM‐loaded nanoconstructs. The feasibility and potential of these newly developed architectures for designing carrier systems for GEM to achieve augmented prognosis for pancreatic cancer are reported.
The focus of regenerative therapies is to replace or enrich diseased or injured cells and tissue in an attempt to replenish the local environment and function, while slowing or halting further degeneration. Targeting neurological diseases specifically is difficult, due to the complex nature of the central nervous system, including the difficulty of bypassing the brain's natural defense systems. While cell-based regenerative therapies show promise in select tissues, preclinical and clinical studies have been largely unable to transfer these successes to the brain. Advancements in nanotechnologies have provided new methods of central nervous system access, drug and cell delivery, as well as new systems of cell maintenance and support that may bridge the gap between regenerative therapies and the brain. In this review, we discuss current regenerative therapies for neurological diseases, nanotechnology as nanocarriers, and the technical, manufacturing, and regulatory challenges that arise from inception to formulation of nanoparticle-regenerative therapies.
A new class of biobased nanocarriers—Soysomes—have been discovered and investigated. These nanocarriers are derived from a synthetically accessible, scalable macromolecule, methoxylated sucrose soyate polyol (MSSP), derived from chemical building blocks obtained from soybean oil and sucrose. We observed for the first time that MSSP, when dissolved in an organic solvent of different polarity and slowly added to an aqueous phase at a predetermined rate under ‘nanoprecipitation’ conditions, will form a stable, self-assembled structure with a size range of 100-200 nm depending on the polarity difference between the precipitating solvent-pairs. Without the aid of poly (ethylene glycol) or of any surfactants, these Soysomes were found to be stable in water for an extended period and can withstand the destabilizing effect of time, temperature and pH. We also found that Soysomes were able to encapsulate and release hydrophobic bioactive compound, such as curcumin. Both MSSP and their self-assembled structures were highly biocompatible, and did not trigger cellular toxicity to mammalian cell lines. Our experiments showed that such 100% biobased, non-cytotoxic material as MSSP and related class of products have the potential for use towards the sustainable manufacturing of drug nanocarriers for biomedical applications.
An esterase-sensitive glutathione persulfide (GSSH) donor (BW-GP-401) is described. The release profile was studied by monitoring the formation of lactone and direct trapping of GSSH with 1-fluoro-2,4-dinitrobenzene (DNFB). The donor was examined for its inhibitory effect toward glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Under highly oxidative conditions, the donor also shows cytoprotective effects in H9c2 cardiomyocytes.
Neurotherapeutics for the treatment of central nervous system (CNS) disorders must overcome challenges relating to the blood-brain barrier (BBB), brain tissue penetration, and the targeting of specific cells. Neuroinflammation mediated by activated microglia is a major hallmark of several neurological disorders, making these cells a desirable therapeutic target. Building on the promise of hydroxyl-terminated generation four polyamidoamine (PAMAM) dendrimers (D4-OH) for penetrating the injured BBB and targeting activated glia, we explored if conjugation of targeting ligands would enhance and modify brain and organ uptake. Since mannose receptors [cluster of differentiation (CD) 206] are typically over-expressed on injured microglia, we conjugated mannose to the surface of multifunctional D4-OH using highly efficient, atom-economical, and orthogonal Cu(I)-catalyzed alkyne-azide cycloaddition (CuAAC) click chemistry and evaluated the effect of mannose conjugation on the specific cell uptake of targeted and non-targeted dendrimers both in vitro and in vivo. In vitro results indicate that the conjugation of mannose as a targeting ligand significantly changes the mechanism of dendrimer internalization, giving mannosylated dendrimer a preference for mannose receptor-mediated endocytosis as opposed to non-specific fluid phase endocytosis. We further investigated the brain uptake and biodistribution of targeted and non-targeted fluorescently labeled dendrimers in a maternal intrauterine inflammation-induced cerebral palsy (CP) rabbit model using quantification methods based on fluorescence spectroscopy and confocal microscopy. We found that the conjugation of mannose modified the distribution of D4-OH throughout the body in this neonatal rabbit CP model without lowering the amount of dendrimer delivered to injured glia in the brain, even though significantly higher glial uptake was not observed in this model. Mannose conjugation to the dendrimer modifies the dendrimer's interaction with cells, but does not minimize its inherent inflammation-targeting abilities.
Polymer-drug conjugates have been intensely studied in the context of improving cancer chemotherapy and yet the only polymer-drug conjugate on the market (Movantik®) has a different therapeutic application (relieving opioid-induced constipation). In parallel, a number of studies have recently been published proposing the use of this approach for treating diseases other than cancer. In this commentary, we analyse the many and very diverse applications that have been proposed for polymer-drug conjugates (ranging from inflammation to cardiovascular diseases) and the rationales underpinning them. We also highlight key design features to be considered when applying polymer-drug conjugates to these new therapeutic areas.
In this report, poly(amide amine) (PAMAM) dendrimer and Heparin-grafted-monomethoxy polyethylene glycol (HEP-mPEG) were synthesized and characterized. In aqueous solution, the generation 4 PAMAM dendrimers (G4.0-PAMAM) existed as nanoparticles with the particle size 5.63 nm. However, after electrostatic complexation with HEP-mPEG to form a core@shell structure G4.0-PAMAM@HEP-mPEG, the size of nanoparticles was significantly increased (73.82 nm). The G4.0-PAMAM@HEP-mPEG nanoparticles showed their ability to effectively encapsulate doxorubicin (DOX) for prolonged and controlled release. The cytocompatibility of G4.0-PAMAM@HEP-mPEG nanocarriers was significantly increased compared with its parentally G4.0-PAMAM dendrimer in both mouse fibroblast NIH3T3 and the human tumor HeLa cell lines. DOX was effectively encapsulated into G4.0-PAMAM@HEP-mPEG nanoparticles to form DOX-loaded nanocarriers (DOX/G4.0-PAMAM@HEP-mPEG) with high loading efficiency (73.2%). The release of DOX from DOX/G4.0-PAMAM@HEP-mPEG nanocarriers was controlled and prolonged up to 96 h compared with less than 24 h from their parentally G4.0-PAMAM nanocarriers. Importantly, the released DOX retained its bioactivity by inhibiting the proliferation of monolayer-cultured cancer HeLa cells with the same degree of fresh DOX. This prepared G4.0-PAMAM@HEP-mPEG nanocarrier can be a potential candidate for drug delivery systems with high loading capacity and low systemic toxicity in cancer therapy.
N-Acetylcysteine was given intravenously and as three fast dissolving and one slow-release formulation, on separate occasions, as a single dose of 600 mg to 10 fasting (5 men and 5 women) healthy volunteers. Blood and urine were sampled for the following 12 h. Renal clearance constituted around 30% of total body clearance, which was 0.21 l/h/kg. Volume of distribution was 0.33 l/kg, consistent with distribution mainly to extracellular water. The late elimination half-life was 2.27 h and the mean residence time 1.62 h. The slow-release tablet resulted in a flattened plasma concentration-time curve typical of slow release formulations, while the other three oral formulations were rapidly absorbed. The oral availability of N-acetylcysteine varied between 6 and 10%, with the slow-release tablet having the lowest and the fast dissolving tablet the highest availability.
In the present study we tested whether N-acetyl-L-cysteine (LNAC) affects apoptotic death of neuronal cells caused by trophic factor deprivation. LNAC, an antioxidant, elevates intracellular levels of glutathione. We used serum-deprived PC12 cells, neuronally differentiated PC12 cells deprived of serum and NGF, and NGF-deprived neonatal sympathetic neurons. In each case LNAC prevents apoptotic DNA fragmentation and maintains long-term survival in the absence of other trophic support. Unlike NGF, LNAC does not induce or maintain neurite outgrowth or somatic hypertrophy. To rule out actions of LNAC metabolic derivatives, we assessed N-acetyl-D-cysteine (DNAC). DNAC also prevents death of PC12 cells and sympathetic neurons. However, other antioxidants were ineffective in this regard. Since it has been hypothesized that trophic factors prevent neuronal death by either preventing or coordinating cell cycle progression, we tested whether LNAC or DNAC treatment can affect cell cycle. We found that both (but not other antioxidants) suppress proliferation and DNA synthesis by PC12 cells and do so at concentrations similar to those at which they prevent apoptotic death. Although the abilities of LNAC and DNAC to rescue cells from apoptosis triggered by trophic factor deprivation could derive from their direct influences on cellular responsiveness to oxidative stress, our observations raise the possibility of a mechanism involving cell cycle regulation.
To determine the therapeutic effect of sulfur amino acid supplementation in HIV infection we randomized 40 patients with antiretroviral therapy (ART; study 1) and 29 patients without ART (study 2) to treatment for 7 months with N-acetyl-cysteine or placebo at an individually adjusted dose according to a defined scheme. The main outcome measures were the change in immunological parameters including natural killer (NK) cell and T cell functions and the viral load. Both studies showed consistently that N-acetyl-cysteine causes a marked increase in immunological functions and plasma albumin concentrations. The effect of N-acetyl-cysteine on the viral load, in contrast, was not consistent and may warrant further studies. Our findings suggest that the impairment of immunological functions in HIV+ patients results at least partly from cysteine deficiency. Because immune reconstitution is a widely accepted aim of HIV treatment, N-acetyl-cysteine treatment may be recommended for patients with and without ART. Our previous report on the massive loss of sulfur in HIV-infected subjects and the present demonstration of the immunoreconstituting effect of cysteine supplementation indicate that the HIV-induced cysteine depletion is a novel mechanism by which a virus destroys the immune defense of the host and escapes immune elimination.
Radiographic contrast agents can cause a reduction in renal function that may be due to reactive oxygen species. Whether the reduction can be prevented by the administration of antioxidants is unknown.
We prospectively studied 83 patients with chronic renal insufficiency (mean [+/-SD] serum creatinine concentration, 2.4+/-1.3 mg per deciliter [216+/-116 micromol per liter]) who were undergoing computed tomography with a nonionic, low-osmolality contrast agent. Patients were randomly assigned either to receive the antioxidant acetylcysteine (600 mg orally twice daily) and 0.45 percent saline intravenously, before and after administration of the contrast agent, or to receive placebo and saline.
Ten of the 83 patients (12 percent) had an increase of at least 0.5 mg per deciliter (44 micromol per liter) in the serum creatinine concentration 48 hours after administration of the contrast agent: 1 of the 41 patients in the acetylcysteine group (2 percent) and 9 of the 42 patients in the control group (21 percent; P=0.01; relative risk, 0.1; 95 percent confidence interval, 0.02 to 0.9). In the acetylcysteine group, the mean serum creatinine concentration decreased significantly (P<0.001), from 2.5+/-1.3 to 2.1+/-1.3 mg per deciliter (220+/-118 to 186+/-112 micromol per liter) 48 hours after the administration of the contrast medium, whereas in the control group, the mean serum creatinine concentration increased nonsignificantly (P=0.18), from 2.4+/-1.3 to 2.6+/-1.5 mg per deciliter (212+/-114 to 226+/-133 micromol per liter) (P<0.001 for the comparison between groups).
Prophylactic oral administration of the antioxidant acetylcysteine, along with hydration, prevents the reduction in renal function induced by contrast agents in patients with chronic renal insufficiency.
Target-specific DNA delivery requires vectors that combine stability in the biological milieu, receptor-mediated uptake into target cells, and intracellular activation to mediate transgene expression. This is achieved here using polymer-coated vectors based on plasmid DNA complexed with a reductively degradable polycation (RPC), designed for intercellular degradation. The RPC were prepared by oxidation of the terminal cysteinyl thiol groups of Cys(Lys)10Cys. The complexes were coated and surface-cross-linked using multivalent reactive copolymers of N-(2-hydroxypropyl)methacrylamide (PHPMA), providing a unique combination of steric and reversible lateral stabilization, known to promote extended circulation in the bloodstream. Coated complexes containing RPC exhibited lateral stabilization that was reversible by treatment with 2.5 mM dithiothreitol, releasing free DNA after incubation with a polyanion. In contrast, coated complexes containing nonreducible poly(l-lysine) (PLL) were not destabilized by reduction. The biological usefulness of this trigger mechanism was examined by measuring transfection activity in human retinoblast 911 cells of coated complexes, based on PLL or RPC, targeted to cell surface receptors by covalent linkage of basic fibroblast growth factor. The levels of transgene expression observed for RPC-based targeted vectors indicated efficient intracellular activation, authenticating the concept that lateral stabilization introduced by surface coating with PHPMA can be reversed by intracellular reduction.
Intrauterine and maternal systemic infections are proposed causes of preterm labor. The resulting prematurity is associated with 75% of infant mortality and 50% of long-term neurologic handicaps. We hypothesize that free radicals generated in large quantities during an inflammatory response shift the fetomaternal redox balance to an oxidative state, compromising the fetus. Thus, if our working hypothesis is correct, selective inactivation of free radicals with N-acetylcysteine (NAC), an antioxidant and glutathione (GSH) precursor, would improve the outcome of preterm deliveries associated with inflammation. We tested aspects of this hypothesis in an animal model of preterm labor and fetal damage (death).
NAC (1 g/kg) was administered orally to C57Bl/6 mice injected intraperitoneally with either 10 microg lipopolysaccharide (LPS) or saline solution (CRL) on day 16 of gestation. The latency period (time from injection to delivery of the first pup) and fetal viability were recorded. To discriminate between an effect of prematurity from an effect of inflammation, and to document any improvement in survival, mice were killed at 3, 6, and 16 hours after injection. Maternal and fetal redox states were approximated by measuring hepatic GSH.
Each C57Bl/6 LPS-treated mouse delivered prematurely after a significantly shorter latency period (LPS: 16.8 hours [95% CI 15.9-17.6] vs CRL: 54.7 hours [95% CI 43.8-65.5]). NAC doubled the latency interval of LPS-treated animals to 35.2 hours (95% CI 21.0-49.2). LPS alone resulted in a 100% rate of stillbirth. Fifty-eight percent of fetuses were already dead 16 hours after LPS. In contrast, only 33% of fetuses were dead 16 hours after LPS (P =.001) when NAC was given. LPS was followed by a reduction in maternal (LPS: 26.3 nmol/mg [95% CI 19.9-32.8] vs CRL: 41.3 nmol/mg [95% CI 34.7-47.9, P <.01]) and fetal GSH (LPS: 19.7 nmol/mg [95% CI 11.7-27.8] vs CRL: 34.5 nmol/mg [95% CI 32.0-37.0, P <.001]). This decline was reversed by NAC (NAC/LPS maternal GSH: 37.0 nmol/mg [95% CI 22.5-51.5] and fetal GSH: 28.4 nmol/mg [95% CI 22.8-33.9]). Importantly, maternal liver GSH impacted on fetal survival. NAC/LPS mothers with living pups 16 hours after LPS had significantly higher liver GSH compared with NAC/LPS mothers whose pups died in utero. In fact, all NAC-treated mice whose hepatic GSH exceeded 20 nmol/mg had living fetuses at 16 hours.
Maternal inflammation in C57Bl/6 mice results in oxidative stress associated with maternal and fetal GSH depletion. Oxidative stress damages the fetus independent of prematurity. Restoration of maternal and fetal oxidative balance by NAC protects the fetus and reduces the rate of preterm birth.
Dendrimers are branched, synthetic polymers with layered architectures that show promise in several biomedical applications. By regulating dendrimer synthesis, it is possible to precisely manipulate both their molecular weight and chemical composition, thereby allowing predictable tuning of their biocompatibility and pharmacokinetics. Advances in our understanding of the role of molecular weight and architecture on the in vivo behavior of dendrimers, together with recent progress in the design of biodegradable chemistries, has enabled the application of these branched polymers as anti-viral drugs, tissue repair scaffolds, targeted carriers of chemotherapeutics and optical oxygen sensors. Before such products can reach the market, however, the field must not only address the cost of manufacture and quality control of pharmaceutical-grade materials, but also assess the long-term human and environmental health consequences of dendrimer exposure in vivo.
The design, synthesis and characterization of a series of zero generation (G0) PAMAM dendrimer-based prodrugs for the potential enhancement of drug solubility and bioavailability are described. Naproxen, a poorly water-soluble drug, was conjugated to dendrimers either directly by an amide bond or by ester bonds using either L-lactic acid or diethylene glycol as a linker. All of the prodrugs were more hydrophilic than the parent drug, as evaluated by drug partitioning between 1-octanol and phosphate buffer (pH 7.4). Hydrolysis of the conjugates was measured at 37 degrees C in hydrochloric acid buffer (pH 1.2), phosphate buffer (pH 7.4), borate buffer (pH 8.5) and in 80% human plasma. The amide conjugate and both ester conjugates were chemically stable at all pHs over 48 h of incubation. Naproxen was enzymatically released from both ester conjugates in plasma; the lactic ester conjugate hydrolyzed slowly with only 25% of naproxen released after 24h, the diethylene glycol ester conjugate cleaved rapidly following pseudo first order kinetics (t(1/2) = 51 min). G0 PAMAM dendrimer prodrugs with an appropriate linker (diethylene glycol) show good potential as carriers for oral delivery.
A comparative species investigation of the relative pharmacologic effects of sulfur amino acids was conducted using young chicks, rats, and pigs. Ingestion of excess Met, Cys, or Cys-Cys supplemented at 2.5-, 5.0-, 7.5-, or 10 times the dietary requirement in a corn-soybean meal diet depressed chick growth to varying degrees. Strikingly, ingestion of excess Cys at 30 g/kg Cys (7.5-times the dietary requirement) caused a chick mortality rate of 50% after only 5 d of feeding. Growth was restored and chick mortality was reduced by supplementing diets containing 25 g/kg excess Cys with KHCO3 at 10 g/kg. Additionally, mortality was prevented by supplementing the drinking water of chicks receiving 25 g/kg supplemental Cys with H2O2 (0.05% final concentration). After young rats and pigs consumed excess Cys or Cys-Cys up to 40 g/kg for 14 d, weight gain was severely depressed, but we observed no mortality. An excess of dietary Cys-Cys>or=48 g/kg caused some mortality in rats. Pigs exhibited rapid recovery from growth-depressing excesses of Cys or Cys-Cys. These results lend credence to the acute toxic effects associated with the ingestion of excess sulfur amino acids and highlight the potential for excess dietary cyst(e)ine to be more pernicious than Met in certain species.
Relative bioavailability and toxicity of N-acetyl-l-Cys (NAC) were evaluated in 9-d chick growth assays. The bioavailability of NAC relative to Cys was determined by feeding young chicks a highly purified crystalline AA diet singly deficient in Cys. Bio-availability estimates were obtained using standard slope-ratio methodology. N-Acetyl-l-cysteine was shown to be as effective as Cys in supporting chick growth, and was assigned a relative bioavailability value of 100%. To assess toxicity, a nutritionally adequate corn-soybean meal diet was supplemented with graded concentrations of NAC (isomolar to 10, 20, 30, or 40 g/kg of Cys, as-fed). When NAC supplied 10 or 20 g/kg of Cys, chick growth performance was unaffected, but NAC supplying 30 or 40 g/kg of Cys reduced (P < 0.05) BW gain by 13 and 34%, respectively, relative to the unsupplemented control diet. Only plasma-free NAC was substantially increased (P < 0.05) because of excess dietary NAC; plasma-free Cys was unaltered. We concluded that dietary NAC is efficacious in supplying Cys in support of chick growth, and only large excesses of NAC are growth depressing. Hence, the human clinical benefits of oral NAC likely result from its ability to deliver Cys safely and effectively to the portal circulation.
NO transfer reactions between protein and peptide cysteines have been proposed to represent regulated signaling processes. We used the pharmaceutical antioxidant N-acetylcysteine (NAC) as a bait reactant to measure NO transfer reactions in blood and to study the vascular effects of these reactions in vivo. NAC was converted to S-nitroso-N-acetylcysteine (SNOAC), decreasing erythrocytic S-nitrosothiol content, both during whole-blood deoxygenation ex vivo and during a 3-week protocol in which mice received high-dose NAC in vivo. Strikingly, the NAC-treated mice developed pulmonary arterial hypertension (PAH) that mimicked the effects of chronic hypoxia. Moreover, systemic SNOAC administration recapitulated effects of both NAC and hypoxia. eNOS-deficient mice were protected from the effects of NAC but not SNOAC, suggesting that conversion of NAC to SNOAC was necessary for the development of PAH. These data reveal an unanticipated adverse effect of chronic NAC administration and introduce a new animal model of PAH. Moreover, evidence that conversion of NAC to SNOAC during blood deoxygenation is necessary for the development of PAH in this model challenges conventional views of oxygen sensing and of NO signaling.
We have previously demonstrated that transferrin-polycation conjugates are efficient carrier molecules for the introduction of genes into eukaryotic cells. We describe here a more specific method for conjugation of transferrin with DNA-binding compounds involving attachment at the transferrin carbohydrate moiety. We used the polycation poly(L-lysine) or the DNA intercalator, ethidium homodimer as DNA-binding domains. Successful transferrin-receptor-mediated delivery and expression of the Photinus pyralis luciferase gene in K562 cells has been shown with these new transferrin conjugates. The activity of the transferrin-ethidium homodimer (TfEtD) conjugates is low relative to transferrin-polylysine conjugates; probably because of incomplete condensation of the DNA. However, DNA delivery with TfEtD is drastically improved when ternary complexes of the DNA with TfEtD and the DNA condensing agent polylysine are prepared. The gene delivery with the carbohydrate-linked transferrin-polylysine conjugates is equal or superior to described conjugates containing disulfide linkage. The new ligation method facilitates the synthesis of large quantities (greater than 100 mg) of conjugates.
Data on the synthesis, physicochemical characterisation and in vitro and in vivo biological properties of the new, nontargeted or antibody-targeted polymer–doxorubicin conjugates designed as anticancer drugs are presented. In the conjugates, the anticancer drug doxorubicin (DOX) is attached to the polymer carrier via a simple hydrolytically labile spacer containing either a hydrazone bond or cis-aconitic acid residue. In vitro incubation of the conjugates in buffers led to a fast DOX release from the polymer at pH 5 (modelling intracellular environment) while at pH 7.4 (modelling blood) the conjugates are relatively stable. Cytotoxicity of the conjugates to T cell lymphoma EL4 depended on the detailed structure of the spacer and the method used for antibody attachment and was much higher compared with the effect of similar classic conjugates (DOX attached to the polymer via enzymatically degradable spacer). In both protective and therapeutic regimes of drug administration, the in vivo anti-tumor activity of the hydrazone conjugates containing only DOX was significantly enhanced (T cell lymphoma EL4, C57BL/10 mice) in comparison with free DOX or classic PK1, the PHPMA–DOX conjugate clinically tested at present. Increasing the molecular weight of the polymer carrier resulted in a more pronounced in vivo antitumor effect. Antibody-targeted conjugates with DOX bound via hydrazone bond exhibited even more extensive inhibition of the tumor growth with some long-term survivors. No survivors were observed after treatment of mice with free DOX or the nontargeted PHPMA–DOX conjugate.
Most pharmaceutical and gene therapy applications of targeted liposomes presently suffer from inefficient contents delivery to the cytoplasm of target cells. We report a plasma-stable liposome, composed of synthetic, naturally occurring diplasmenylcholine (1,2-di-O-(Z-1‘-hexadecenyl)-sn-glycero-3-phosphocholine; DPPlsC), that rapidly and efficiently releases its contents at endosomal pHs. Acid-catalyzed hydrolysis of these liposomes produces glycerophosphocholine and fatty aldehydes, leading to greatly enhanced liposome permeability (t50% release 1−4 h between pH 4.5−5.5) when >20% of the vinyl ether lipid has been hydrolyzed. Plasma stability of nonhydrolyzed 9:1 DPPlsC/dihydrocholesterol liposomes exceeds 48 h at 37 °C, pH 7.4 in 50% serum; pure DPPlsC liposomes remain stable in 10% serum under the same conditions. Fluorescence assays of KB cells treated with 99.5:0.5 DPPlsC/DSPE−PEG3350−folate liposomes containing encapsulated propidium iodide (PI) indicate that 83% of the PI escapes the endosomal compartment within 8 h to produce intensely stained nucleii. The IC50 value of 1-β-arabinofuranosylcytosine (Ara-C) encapsulated in DPPlsC/DSPE−PEG3350−folate liposomes is 0.49 μM in KB cell cultures, a 6000-fold enhancement in cytotoxicity compared with free drug (2.8 mM). Empty DPPlsC/DSPE−PEG3350−folate liposomes had no effect on DNA synthesis, indicating that DPPlsC and its degradation products are benign to cell function at these lipid concentrations. Our results suggest that concurrent application of selective targeting and membrane translocation mechanisms in drug carriers can significantly increase their efficacy.
Glutathione (GSH) is important in in vitro models of radiation and drug resistance, but its clinical relevance is uncertain. GSH levels were measured prospectively in 35 patients with breast cancer to establish normal ranges and to determine whether differences exist between tumor, lymph node metastases (when present), and normal breast tissue.
Fresh tissue was obtained immediately from discarded surgical specimens, and total GSH levels were measured with the Tietze cyclic reduction assay. When possible, multiple sites were assayed in each tumor to assess intratumor variability.
GSH levels in primary breast tumors were more than twice the levels found in normal breast tissue, and levels in lymph node metastases were more than four times the levels in normal breast tissue. The levels of GSH in normal lymph nodes were similar to levels in normal breast tissue. GSH levels in different areas of the same breast tumor ranged from below those of normal breast tissue up to 11 times normal tissue levels. No correlation was found between tumor GSH levels and common clinical parameters such as tumor size, nodal status, stage, estrogen receptor levels, or progesterone receptor levels.
GSH appears to be a marker of breast malignancy that is independent of hormonal receptor status and stage and may be a marker of cells with increased potential for dissemination. Because of tumor heterogeneity, multiple sites should be sampled whenever possible. Long-term clinical follow-up will be necessary to ascertain the usefulness of tumor and lymph node GSH levels in predicting prognosis.
Methylmercury is a ubiquitous environmental pollutant and potent neurotoxin. Treatment of methylmercury poisoning relies almost exclusively on the use of chelating agents to accelerate excretion of the metal. The present study demonstrates that oral administration of N-acetylcysteine (NAC), a widely available and largely nontoxic amino acid derivative, produces a profound acceleration of urinary methylmercury excretion in mice. Mice that received NAC in the drinking water (10 mg/ml) starting at 48 hr after methylmercury administration excreted from 47 to 54% of the 203Hg in urine over the subsequent 48 hr, as compared to 4-10% excretion in control animals. When NAC-containing water was given from the time of methylmercury administration, it was even more effective at enhancing urinary methylmercury excretion and at lowering tissue mercury levels. In contrast, excretion of inorganic mercury was not affected by oral NAC administration. The ability of NAC to enhance methylmercury excretion when given orally, its relatively low toxicity, and is wide availability in the clinical setting indicate that it may be an ideal therapeutic agent for use in methylmercury poisoning.
Liposomes containing antimonial compounds trapped in the aqueous phase were tested in the treatment of experimental leishmaniasis. The rationale of this approach was based on the hypothesis that the liposomes and the parasite are taken up by the same cell, the reticuloendothelial cell, and we present electron microscopic evidence that supports this hypothesis. Suppression of leishmaniasis was quantified by determining the total number of parasites per liver from impression smears. When two antimonials, meglumine antimoniate and sodium stibogluconate, were encapsulated within liposomes, each was more than 700 times more active compared to either of the free (unencapsulated) drugs. After infection, if untreated, all of the hamsters eventually would die from the disease. Liposome-encapsulated meglumine antimoniate was about 330-640 times more effective in causing a drop in the death rate than was the free antimonial. The efficacy of treatment was influenced by the lipid composition and charge of the liposomes. For example, positively charged liposomes containing egg phosphatidylcholine were much less effective than negatively charged ones. In contrast, positively and negatively charged sphingomyelin liposomes were equally effective. Liposomes containing phosphatidylserine (which were negatively charged, but also had a much higher charge density) were among the less-effective preparations. Among those tested, the most consistently efficacious liposomes contained highly saturated long-chain phospholipids (eg., dipalmitoyl phosphatidylcholine), cholesterol, and a negative charge.
We conclude that liposomes may be useful as carriers of drugs to treat infectious diseases involving the reticuloendothelial system. The toxicities of antimony are very similar to those of arsenic. Encapsulation of antimonial drugs and reduction of the dose required for effective therapy should minimize such systemic toxicities as acute cardiomyopathy and toxic nephritis.
Free radicals may play a role in the pathogenesis of amyotrophic lateral sclerosis.
To investigate the efficacy of the free radical scavenging agent acetylcysteine in patients with amyotrophic lateral sclerosis.
Randomized, double-blind, placebo-controlled clinical trial to assess the effect of treatment with acetylcysteine on survival and disease progression.
A university hospital referral setting.
One hundred ten consecutive patients who fulfilled the diagnostic criteria for amyotrophic lateral sclerosis, followed up at monthly intervals for 12 months.
Acetylcysteine or placebo in a dose of 50 mg/kg per day subcutaneously for 12 months.
Survival.
After 12 months, 35 patients (65%) treated with acetylcysteine and 30 (54%) given placebo were still alive (hazard ratio, 0.74 in the acetylcysteine group relative to the placebo group; 95% confidence interval, 0.41 to 1.33; log-rank test, P = .31). Rates of disease progression, as expressed by decline in muscle strength, pulmonary function, disability, and bulbar function were similar in both groups. In the subgroup of 81 patients with limb onset of the disease, 28 patients (74%) in the acetylcysteine group and 22 (51%) in the placebo group survived 12 months (hazard ratio, 0.50; 95% confidence interval, 0.24 to 1.04; P = .06). In the bulbar subgroup of 29 patients, seven patients (44%) receiving acetylcysteine and eight (62%) receiving placebo were alive at the end of the study (hazard ratio, 1.66; 95% confidence interval, 0.56 to 4.99; P = .36).
In this trial, treatment with the free radical scavenger acetylcysteine did not result in a major increase in 12-month survival or a reduction in disease progression in patients with amyotrophic lateral sclerosis.
Disulfide-stabilized Fv (dsFv)-immunotoxins are recombinant immunotoxins in which the inherently unstable Fv moiety, composed of the VH-VL heterodimer, is stabilized by a disulfide bond engineered between structurally conserved framework positions of VH and VL. Anti-Tac(dsFv)-PE38KDEL is composed of such a dsFv, directed to the alpha subunit of the IL2 receptor (IL2R), and containing a truncated form of Pseudomonas exotoxin (PE38KDEL). We have found this new type of immunotoxin to be indistinguishable in its in vitro activity and specificity from its single-chain immunotoxin counterpart, anti-Tac(Fv)-PE38KDEL. We have now examined the therapeutically relevant factors, including stability, pharmacokinetics, and antitumor activity of this new disulfide-stabilized Fv-immunotoxin. We found that anti-Tac(dsFv)-PE38KDEL was specifically cytotoxic to human activated T-lymphocytes in addition to IL2R bearing cell lines. Anti-Tac(dsFv)-PE38KDEL was considerably more stable at 37 degrees C in human serum and in buffered saline than the single-chain immunotoxin, anti-Tac(Fv)-PE38KDEL. The half-life in blood was similar for both immunotoxins (approx. 20 min). The therapeutic potential of the disulfide-stabilized immunotoxin was evaluated using an animal model of immunodeficient mice bearing subcutaneous tumor xenografts of human IL2R-bearing cells. Anti-Tac(dsFv)-PE38KDEL caused complete regression of tumors with no toxic effects in mice. Because dsFv-immunotoxins are more stable and can be produced with significantly improved yields compared to scFv-immunotoxins, dsFv-immunotoxin may be more useful for therapeutic applications.
The calicheamicin family of antitumor antibiotics are capable of producing double-stranded DNA breaks at sub-picomolar concentrations. Their potency suggested that the calicheamicins would be excellent candidates for targeted delivery and a hydrazide prepared from the most potent and abundant of the naturally occurring derivative, gamma 1I, was linked to oxidized sugars on CT-M-01, an internalizing anti-polyepithelial mucin antibody. The conjugates retained the immunoreactivity of the unmodified antibody and were specifically cytotoxic toward antigen positive tumor cells in vitro and in vivo. Hydrazide analogues of less potent calicheamicin derivatives were also prepared and conjugated to CT-M-01. Comparison of the therapeutic efficacy of the conjugates against the MX-1 xenograft tumor implanted s.c. in nude mice showed that conjugates of derivatives missing the rhamnose, a sugar residue that is part of the DNA binding region of the drug, were not as promising as antitumor therapies. However, conjugates of two derivatives, alpha 3I and N-acetyl-gamma 1I, in which the rhamnose residue is present but the amino sugar residue of the parent drug is either missing or modified, significantly inhibited tumor growth over a 4-fold dose range and produced long-term tumor-free survivors. Sterically hindering methyl groups adjacent to the disulfide in the linker further increased the therapeutic window of these potent conjugates.
Antisense oligonucleotides have been covalently attached to asialoglycoprotein (ASGP) via disulfide bond conjugation chemistry. These conjugates were characterized extensively by an array of chemical, chromatographic, and spectroscopic means. Multiple (approximately six) oligonucleotides can be conjugated to each ASGP molecule. The molecular conjugates were used to deliver antisense oligonucleotides complementary to the mRNA of the interleukin 6 signal transduction protein (gp130) to modulate the acute phase response of hepatoma (HepG2) cells in vitro. These conjugates were biologically active, as measured by inhibition of the cytokine-stimulated up-regulation of the acute phase protein haptoglobin. The level of inhibition was comparable to that found with previous technology featuring noncovalent complexes of ASGP-poly(L-lysine) and oligonucleotide. Because of the ability to control the stoichiometry of the conjugate and its unimolecular nature (as opposed to bimolecular for the noncovalent conjugates), this methodology holds great promise for further development and application.
The treatment of chronic obstructive pulmonary disease (COPD) with inhaled corticosteroids or anti-oxidants is still under debate and the identification of sub-groups of COPD patients who may benefit from either anti-inflammatory or anti-oxidant treatment is needed. We re-analysed data from an earlier study of inhaled beclomethasone therapy in COPD (n = 28) and asthma (n = 28) patients in order to determine patient characteristics that predict a favourable inhaled steroid treatment effect. A higher bronchodilatory response, a faster decline of FEV1 prior to the treatment period and a lower Tiffeneau index were significantly related to more beneficial treatment effects. Increased smoking tended to be related to less steroid treatment benefits, though it was not statistically significant. In this paper these findings are presented in light of the available literature on anti-inflammatory and anti-oxidant COPD treatment. On this basis the hypothesis is presented that anti-oxidant treatment might be relatively more effective among those COPD patients who respond less well to inhaled steroids (low reversibility and heavy smoking).
This investigation is part of an effort to develop chemoprevention for carcinogenesis of the large bowel. The agent investigated is N-acetylcysteine (NAC). We used as a predictive biomarker, the proliferative index (PI), in a short-term human study. Patients with previous adenomatous colonic polyps are a cohort with increased risk for colon cancer and an increased PI of colonic crypts. They were randomly assigned to an experimental group given 800 mg/day of NAC for 12 weeks or a placebo group. Using proliferative cell nuclear antigen immunostaining, the PI of colonic crypts was measured prior to and after the treatments. The PI of the NAC group was decreased significantly (P < 0.02) while the placebo group showed no difference (P > 0.45). Since this decrease in PI may be an indicator of decreased risk of colon cancer, more extensive studies of the potential of NAC as a chemopreventive agent for colon cancer appear warranted.
Acute liver failure is a serious condition associated with poor prognosis. It may be associated with changes in systemic hemodynamics, i.e., tissue hypoxia, which contributes to multiple-organ failure. Recent studies have shown that N-acetylcysteine administered to patients with fulminant hepatic failure (paracetamol-induced) increases oxygen delivery and improves survival. The aim of this pilot study was to evaluate N-acetylcysteine administration to patients with non-paracetamol-induced acute liver failure and assess its effect on the clinical course and outcome.
N-acetylcysteine was administered at presentation to 7 patients with non-paracetamol-induced acute liver failure. Patients were followed for changes in clinical parameters (grade of encephalopathy), coagulation factors, biochemical parameters and outcome.
Clinically, 3 patients who initially had grade O/II encephalopathy, did not progress, and have fully recovered. The mean peak prothrombin time, serum factor V, aspartate aminotransferase and alanine aminotransferase levels, all significantly improved. Four patients (57%) have recovered fully (1 patient, although fully recovered, died later from an unrelated cause). Two patients required orthotopic liver transplantation and 1 patient died. N-acetylcysteine administration may have prevented progression to grade III/IV encephalopathy and improved serum coagulation factors. This may account for its beneficial effect on survival in patients who had poor prognostic criteria at base-line. No side effects of the drug were noted.
This study suggests that N-acetylcysteine administration should be considered in all patients with acute liver failure.
High molecular weight polymers (> 20 000 Da) have been widely used as soluble drug carriers to improve drug targeting and therapeutic efficacy. Dendritic polymers are exceptional candidates for the preparation of near monodisperse drug carriers due to their well-defined structure, multivalency, and flexibility for tailored functionalization. We evaluated various dendritic architectures composed of a polyester dendritic scaffold based on the monomer unit 2,2-bis(hydroxymethyl)propanoic acid for their suitability as drug carriers both in vitro and in vivo. These systems are both water soluble and nontoxic. In addition, the potent anticancer drug, doxorubicin, was covalently bound via a hydrazone linkage to a high molecular weight 3-arm poly(ethylene oxide)-dendrimer hybrid. Drug release was a function of pH, and the release rate was more rapid at pH < 6. The cytotoxicity of the DOX-polymer conjugate measured on multiple cancer lines in vitro was reduced but not eliminated, indicating that some active doxorubicin was released from the drug polymer conjugate under physiological conditions. Furthermore, biodistribution experiments show little accumulation of the DOX-polymer conjugate in vital organs, and the serum half-life of doxorubicin attached to an appropriate high molecular weight polymer has been significantly increased when compared to the free drug. Thus, this new macromolecular system exhibits promising characteristics for the development of new polymeric drug carriers.
Covalent binding between N-acetyl-L-cysteine (NAC) and albumin was evaluated kinetically by conducting in vitro experiments.
After 14C-NAC was incubated with human or rat serum, the solution was analyzed by anion-exchange HPLC. The albumin-bound 14C-NAC was quantified by measuring the radioactivity in the albumin fraction.
Ultraviolet chromatograms and/or radiochromatograms indicated the presence of a stable covalent bond between 14C-NAC and either human or rat albumin. By analyzing the time dependence of this protein binding in serum, the first-order binding and dissociation rate constants (k(on) and k(off) were obtained. The serum was treated in a CO2 incubator to avoid oxidative interference, and the initial rates were determined separately. The k(on) values obtained were 0.33 (h(-1)) and 0.48 (h(-1)) for human and rat serum, respectively. L-Cysteine was required to initiate the dissociation of 14C-NAC bound to albumin. Following the addition of appropriate amounts of L-cysteine, the k(off) values were determined to be 0.30-1.0 h(-1) and 0.54-1.4 h(-1) for human and rat serum, respectively.
The k(on) and k(off) values obtained for rat serum were in good agreement with the in vivo plasma protein binding kinetics of NAC in rats, indicating the reliability of this in vitro method for evaluating protein binding. No species differences in protein binding kinetics were found between human and rat serum.
Data on the synthesis, physicochemical characterisation and in vitro and in vivo biological properties of the new, nontargeted or antibody-targeted polymer-doxorubicin conjugates designed as anticancer drugs are presented. In the conjugates, the anticancer drug doxorubicin (DOX) is attached to the polymer carrier via a simple hydrolytically labile spacer containing either a hydrazone bond or cis-aconitic acid residue. In vitro incubation of the conjugates in buffers led to a fast DOX release from the polymer at pH 5 (modelling intracellular environment) while at pH 7.4 (modelling blood) the conjugates are relatively stable. Cytotoxicity of the conjugates to T cell lymphoma EL4 depended on the detailed structure of the spacer and the method used for antibody attachment and was much higher compared with the effect of similar classic conjugates (DOX attached to the polymer via enzymatically degradable spacer). In both protective and therapeutic regimes of drug administration, the in vivo anti-tumor activity of the hydrazone conjugates containing only DOX was significantly enhanced (T cell lymphoma EL4, C57BL/10 mice) in comparison with free DOX or classic PK1, the PHPMA-DOX conjugate clinically tested at present. Increasing the molecular weight of the polymer carrier resulted in a more pronounced in vivo antitumor effect. Antibody-targeted conjugates with DOX bound via hydrazone bond exhibited even more extensive inhibition of the tumor growth with some long-term survivors. No survivors were observed after treatment of mice with free DOX or the nontargeted PHPMA-DOX conjugate.
As we enter the twenty-first century, research at the interface of polymer chemistry and the biomedical sciences has given rise to the first nano-sized (5-100 nm) polymer-based pharmaceuticals, the 'polymer therapeutics'. Polymer therapeutics include rationally designed macromolecular drugs, polymer-drug and polymer-protein conjugates, polymeric micelles containing covalently bound drug, and polyplexes for DNA delivery. The successful clinical application of polymer-protein conjugates, and promising clinical results arising from trials with polymer-anticancer-drug conjugates, bode well for the future design and development of the ever more sophisticated bio-nanotechnologies that are needed to realize the full potential of the post-genomic age.
Positively charged trimethylammonium-functionalized mixed monolayer protected clusters (MMPCs) of different chain lengths (C(8) and C(11)) have been used to bind beta-galactosidase through complementary electrostatic interactions, resulting in complete enzyme inhibition. This inhibition can be reversed in vitro by intracellular concentrations of glutathione (GSH), the main thiol component of the cell. The restoration of activity depends on the chain length of the monolayer. The activity of enzyme bound to particles with C(8) monolayer was completely restored by intracellular concentrations (1-10 mM) of GSH; however, little or no release was observed at extracellular GSH concentrations. In contrast, no restoration was observed for enzyme bound to the C(11) particles at any of the concentrations studied. Taken together, these studies demonstrate that the GSH-mediated release of enzymes bound to MMPCs can be tuned through the structure of the monolayer, a significant tool for protein and drug delivery applications.
The unique properties of dendrimers, such as their high degree of branching, multivalency, globular architecture and well-defined molecular weight, make them promising new scaffolds for drug delivery. In the past decade, research has increased on the design and synthesis of biocompatible dendrimers and their application to many areas of bioscience including drug delivery, immunology and the development of vaccines, antimicrobials and antivirals. Recent progress has been made in the application of biocompatible dendrimers to cancer treatment, including their use as delivery systems for potent anticancer drugs such as cisplatin and doxorubicin, as well as agents for both boron neutron capture therapy and photodynamic therapy.
Stand and deliver! The release of pore-encapsulated fluorescein molecules from a core/shell mesoporous silica nanorod/ superparamagnetic iron oxide nanoparticle carrier in the presence of an external magnetic field and only in response to cell-produced antioxidants indicates promise for such systems in controlled-release drug delivery. (Figure Presented)
Glutathione plays an important role in the antioxidant system that is required for the maintenance of the redox status of the cell, defence against free radicals and detoxification of toxic compounds. Reduced glutathione (redGSH) can be converted to oxidized glutathione (GSSG) during oxidative stress. The ratio of redGSH/total glutathione can be regarded as an index of the redox status and a useful indicator of disease risks. We conducted experiments by the capillary zone electrophoresis method to investigate the alterations of the glutathione status in the blood and tissue samples from patients with breast cancer. The results showed that the levels of redGSH, GSSG, total glutathione and the ratio of redGSH/total glutathione were significantly decreased in the blood of the patients with breast cancer compared to those of the control subjects. The levels of various forms of glutathione were lower and more pronounced in stage III. In contrast, the levels of redGSH, GSSG, total glutathione and the redGSH/total glutathione ratio in breast cancer tissues were significantly increased relative to those of the adjacent cancer-free tissues, especially in stage II. We suggest that the high redGSH levels are associated with the enhancement of cell proliferation and resistance to apoptosis in the cancer cells, and the loss of the large amount of erythrocyte redGSH may be due to increased detoxification capacities and defence against oxidative stress. We propose that redGSH should be regarded as an important biochemical parameter for detecting breast malignancy.
We present a novel functionalization scheme for single-walled carbon nanotubes (SWNTs) to afford nanotube-biomolecule conjugates with the incorporation of cleavable bonds to enable controlled molecular releasing from nanotube surfaces, thus creating "smart" nanomaterials with high potential for chemical and biological applications. With this versatile functionalization, we demonstrate transporting, enzymatic cleaving and releasing of DNA from SWNT transporters, and subsequent nuclear translocation of DNA oligonucleotides in mammalian cells. We further show highly efficient delivery of siRNA by SWNTs and achieving more potent RNAi functionality than a widely used conventional transfection agent. Thus, the novel functionalization of SWNTs with cleavable bonds is highly promising for a wide range of applications including gene and protein therapy.
Dendrimers have unique characteristics including monodispersity and modifiable surface functionality, along with highly defined size and structure. This makes these polymers attractive candidates as carriers in drug delivery applications. Drug delivery can be achieved by coupling a drug to polymer through one of two approaches. Hydrophobic drugs can be complexed within the hydrophobic dendrimer interior to make them water-soluble or drugs can be covalently coupled onto the surface of the dendrimer. Using both methods we compared the efficacy of generation 5 PAMAM dendrimers in the targeted drug delivery of methotrexate coupled to the polymer. The amine-terminated dendrimers bind to negatively charged membranes of cells in a non-specific manner and can cause toxicity in vitro and in vivo. To reduce toxicity and to increase aqueous solubility, modifications were made to the surface hydroxyl groups of the dendrimers. For targeted drug delivery, the dendrimer was modified to have a neutral terminal functionality for use with surface-conjugated folic acid as the targeting agent. The complexation of methotrexate within a dendrimer changes the water insoluble drug into a stable and readily water-soluble compound. When this dendrimer complexed drug, however, was placed in a solution of phosphate buffered saline, the methotrexate was immediately released and displayed diffusion characteristics identical to free methotrexate. Covalently coupled methotrexate dendrimer conjugates were stable under identical conditions in water and buffered saline. Cytotoxicity tests showed that methotrexate as the dendrimer inclusion complex had an activity identical to the free drug in vitro. In contrast, folic acid targeted dendrimer with covalently conjugated methotrexate specifically killed receptor-expressing cells by intracellular delivery of the drug through receptor-mediated endocytosis. This study demonstrates that while drug as a dendrimer inclusion complex is readily released and active in vitro, covalently conjugated drug to dendrimer is better suited for specifically targeted drug delivery.
Maternal infections may induce placental, amniotic and, potentially, fetal inflammatory responses. As cytokine responses may be mediated by oxidative stress, we determined whether the antioxidant N-acetyl-cysteine (NAC), can attenuate maternally induced amniotic and placental cytokine responses to maternal infection (modeled by lipopolysaccharide [LPS]).
Gestation day 18 pregnant rats were (1) treated with LPS (100 microg/kg, body weight; intraperitoneally) alone; (2) pretreated with NAC (300 mg/kg body weight; intraperitoneally) 30 minutes before LPS; (3) posttreated with NAC 120 minutes after LPS; or (4) treated with NAC 30 minutes before and 120 minutes after LPS. Six hours after LPS administration, maternal serum and amniotic fluid interleukin-6 (IL-6) and IL-10 levels, and placental IL-6 messenger RNA levels were determined.
LPS increased maternal serum IL-6 (50 +/- 25 to 3444 +/- 584 pg/mL) and IL-10 (40 +/- 20 to 958 +/- 339 pg/mL) and amniotic fluid IL-6 (59 +/- 25 to 891 +/- 128 pg/mL). Pretreatment and/or posttreatment with NAC attenuated IL-6 in the maternal serum and amniotic fluid and IL-10 in the amniotic fluid. LPS also induced placental IL-6 messenger RNA that was inhibited by treatment with NAC before and after LPS.
NAC inhibition of inflammatory responses may protect the fetus from potential long-term sequelae.
We demonstrate here the effective delivery of a dye payload into cells using 2-nm core gold nanoparticles, with release occurring via place exchange of glutathione onto the particle surface. In vitro experiments demonstrate effective release of drug analogues upon addition of glutathione. Cell culture experiments show rapid uptake of nanoparticle and effective release of payload. The role of glutathione in the release process was demonstrated through improved payload release upon transient increase in glutathione levels achieved via introduction of glutathione ethyl ester into the cell.
Evidence suggests that maternal infections may induce fetal inflammatory responses. Because cytokine actions may be mediated by oxidative stress, we determined whether N-acetylcysteine, an antioxidant, can blunt fetal inflammatory responses to maternal lipopolysaccharide.
Sprague Dawley near-term rats (n = 16) received intraperitoneal lipopolysaccharide (100 microg/kg) at 30 minutes and saline solution or N-acetylcysteine (300 mg/kg) at 150 minutes. An additional group received N-acetylcysteine before and after lipopolysaccharide administration. At 6 hours, rats were killed, and fetal and maternal blood cytokines were determined.
After maternal lipopolysaccharide administration, fetal blood interleukin-6 markedly increased (3 +/- 2 to 1265 +/- 574 pg/mL); N-acetylcysteine that was given before or before and after lipopolysaccharide administration reduced fetal interleukin-6 response to control levels. A similar trend was observed for interleukin-1beta. No effect of N-acetylcysteine on fetal interleukin-10 levels was observed.
Maternal N-acetylcysteine inhibits fetal cytokine responses to maternal lipopolysaccharide, even when given 2 hours after lipopolysaccharide injection. These results suggest that N-acetylcysteine may protect the fetus from sequelae of maternal inflammation.
The safe and efficient delivery of DNA remains the major barrier to the clinical application of non-viral gene therapy. Here, we present novel, biodegradable polymers for gene delivery that are capable of simple graft modification and demonstrate the ability to respond to intracellular conditions. We synthesized poly(beta-amino ester)s using a new amine monomer, 2-(pyridyldithio)-ethylamine (PDA). These cationic, degradable polymers contain pyridyldithio functionalities in the side chains that react with high specificity toward thiol ligands. This reactivity is demonstrated using both mercaptoethylamine (MEA) and the thiol peptide RGDC, a ligand that binds with high affinity to certain integrin receptors. These two polymer derivatives displayed strong DNA binding as determined using electrophoresis and dye exclusion assays. In addition, the MEA-based polymer and plasmid DNA were shown to self-assemble into cationic complexes with effective diameters as low as 100 nm. Furthermore, this DNA binding ability was substantially reduced in response to intracellular glutathione concentrations, which may aid in DNA unpackaging inside the cell. These complexes also displayed low cellular toxicity and were able to mediate transfection at levels comparable to PEI in human hepatocellular carcinoma cells. These results suggest that PDA-based poly(beta-amino ester)s may serve as a modular platform for polymer-mediated gene delivery.
Dendrimer-based prodrugs were used to enhance the transepithelial permeability of naproxen, a low solubility model drug. The stability of the dendrimer-naproxen link was assessed. Naproxen was conjugated to G0 polyamidoamine (PAMAM) dendrimers either by an amide bond or an ester bond. The stability of G0 prodrugs was evaluated in 80% human plasma and 50% rat liver homogenate. The cytotoxicity of conjugates towards Caco-2 cells was determined and the transport of the conjugates across Caco-2 monolayers (37 degrees C) was reported. In addition, one lauroyl chain (L) was attached to the surface group of G0 PAMAM dendrimer of the diethylene glycol ester conjugate (G0-deg-NAP) to enhance permeability. The lactic ester conjugate, G0-lact-NAP, hydrolyzed slowly in 80% human plasma and in 50% rat liver homogenate (t(1/2)=180 min). G0-deg-NAP was hydrolyzed more rapidly in 80% human plasma (t(1/2)=51 min) and was rapidly cleaved in 50% liver homogenate (t(1/2)=4.7 min). The conjugates were non-toxic when exposed to Caco-2 cells for 3h. Permeability studies showed a significant enhancement in the transport of naproxen when conjugated to dendrimers; L-G0-deg-NAP yielding the highest permeability. Dendrimer-based prodrugs with appropriate linkers have potential as carriers for the oral delivery of low solubility drugs such as naproxen.
We have demonstrated glutathione- and cysteine-induced transverse overgrowth on gold nanorods. In aqueous solutions, glutathione and cysteine are preferentially bound to the ends of gold nanorods that are stabilized by a cationic surfactant bilayer. This preferential end binding blocks the growth of the nanorods in the longitudinal direction completely and allows for the growth only in the transverse direction. As a result, the diameters of the nanorods become larger and larger while their lengths remain unchanged, as more and more gold precursor is supplied. In addition, the shape of the nanorods undergoes a gradual change from rods, peanuts, and truncated octahedra to faceted spheres during overgrowth. We believe that this transverse overgrowth provides an alternative means for tailoring the longitudinal plasmon wavelengths and extinction cross sections of gold nanoparticles and will therefore facilitate their use in optics, optoelectronics, and biotechnology.
Reducible polycations represent promising carriers of therapeutic nucleic acids. Oligomers of 2-dimethylaminoethyl methacrylate (DMAEMA) containing terminal thiol groups were synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization using difunctional chain transfer agent. Reducible poly(DMAEMA) (rPDMAEMA) was synthesized by oxidation of the terminal thiol groups, forming a polymer with disulfide bonds in the backbone. Physico-chemical properties of DNA polyplexes of rPDMAEMA were evaluated by dynamic and static light scattering methods, revealing lower structural density and DNA content than control PDMAEMA polyplexes. Cytotoxicity and transfection activity of rPDMAEMA-based DNA polyplexes were evaluated in vitro. In comparison with control PDMAEMA, only minimum toxic effects of rPDMAEMA were observed in a panel of cell lines. Transfection activity was tested in B16F10 mouse melanoma and six human pancreatic cancer cell lines. rPDMAEMA polyplexes showed a comparable or better activity than control PDMAEMA polyplexes.
Peptide nucleic acids (PNAs) are effective antisense reagents that bind specific mRNAs preventing their translation. However, PNAs cannot cross cell membranes, hampering delivery to cells. To overcome this problem we made PNAs membrane-permeant by conjugation to the lipophilic triphenylphosphonium (TPP) cation through a disulphide bond. The TPP cation led to efficient PNA uptake into the cytoplasm where the disulphide bond was reduced, releasing the antisense PNA to block expression of its target gene. This method of directing PNAs into cells is a significant improvement on current procedures and will facilitate in vitro and pharmacological applications of PNAs.
Dendrimers are an emerging group of nanostructured, polymeric biomaterials that have potential as non-viral vehicles for delivering drugs and genetic material to intracellular targets. They have a high charge density with tunable surface functional groups, which can alter the local environment and influence cellular interactions. This can have a significant impact on the intracellular trafficking of dendrimer-based nanodevices. With the help of flow cytometry, fluorescence microscopy, and by using specific inhibitors, the influence of surface functionality on their uptake in A549 lung epithelial cells, and subsequent intracellular distribution was investigated. In this paper, we have shown that even though all the dendrimers are taken up by fluid-phase endocytosis, significant differences in uptake mechanisms exist. Anionic dendrimers appear to be mainly taken up by caveolae mediated endocytosis in A549 lung epithelial cells, while cationic and neutral dendrimers appear to be taken in by a non-clathrin, non-caveolae mediated mechanism that may be by electrostatic interactions or other non-specific fluid-phase endocytosis. These findings open up new possibilities of targeting therapeutic agents to specific cell organelles based on surface charge.
Dendrimer-containing particles for sustained release of com-pounds, Provisional US patent, filed November
- R M Kannan
- R Iezzi
- S Kannan
- B Rajaguru
Kannan R. M., Iezzi, R., Kannan, S., and Rajaguru, B. (2007) Dendrimer-containing particles for sustained release of com-pounds, Provisional US patent, filed November. BC800342D Dendrimer-Drug Conjugates for Intracellular Drug Release Bioconjugate Chem., Vol. 19, No. 12, 2008 2455
N-acetyl cysteine as an antidote in methyl mercury poisoning. Envir
- N Ballatori
Ballatori N, et al. N-acetyl cysteine as an antidote in methyl mercury poisoning. Envir. Health Persp
1998;106(5):267–271.