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Dau release pro fi les of PEG-PLA-Dau micelles and cRGD-PEG-PLA-Dau micelles in pH7.4 and pH6.5 PBS buffer.
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Daunorubicin (Dau) has been widely used in the treatment of many solid tumors. However, the side effects of Dau seriously hinder its further application in clinical use. How to increase the drug accumulation in tumors and reduce the damage to normal tissues is an urgent problem to be solved. In this study, we developed c(RGDfK) decorated PEG-PLA mi...
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... experiments were performed at least three times and all results are expressed as mean § SD (standard deviation). Differences between groups were evaluated by analysis of variance (ANOVA) to demonstrate statistical signi fi cance (P < 0.05). Dau-loading content (DL) and conjugation ef fi ciency (CE) was measured by UV-Vis analysis using a UV-Vis Spectro- photometer. The pre-weighed freeze-dried sample was redissolved in DMSO. By using a pre-established calibra- tion curve, the absorbance of Dau at 480 nm was measured to detect the Dau concentration in the solution and Dau content in the conjugate was calculated to be 5.7 wt%, i.e., on average 88% of PEG-PLA was conjugated with Dau. Attachment of the c(RGDfK) peptide onto HOOC- PEG-b-PLA was con fi rmed by fl uorimetric assay, The molar ratio of the cRGD peptide to PEG-PLA was determined to 96.4%. PEG-PLA-Dau micelles were prepared through solvent evaporation methods. For targeting therapy, the co-assembly method has been proven to be a good choice due to its characteristic of easy to tune content of the targeting moiety. c(RGDfK)-PEG-PLA-Dau micelles were prepared by co-assembling of two amphi- philic block copolymers (PEG-PLA-Dau and cRGD- PEG-PLA). As described by Yutaka Mi (31), 20 wt% RGD could perform good target effect in drug target delivery, in this study we used 20 wt% cRGD to prepare target micelles. Considering the content of cRGD in c (RGDfK)-PEG-PLA was 96.4%,the real RGD content in the micelles was calculated to be 19.3% in c(RGDfK)- PEG-PLA-Dau micelles. The characterizations of PEG- PLA-Dau and cRGD-PEG-PLA-Dau micelles were analyzed by TEM and DLS. As shown in Figure 1, two micelles display spherical structure in TEM experiment, and the mean diameters of PEG-PLA-Dau micelle and cRGD-PEG-PLA-Dau micelle were 43 nm and 52 nm, respectively. The increase of micelle diameter could be interpreted as the decoration of cRGD peptides. The Dau release of PEG-PLA-Dau and cRGD-PEG- PLA-Dau micelles in vitro was examined via dialysis method. As shown in Figure 2, PEG-PLA-Dau micelles displayed sustained-release in pH 7.4 and pH 6.5 PBS buffers. After 120 h of release, more than 50% of Dau had been released from micelles in pH 6.5 PBS, while only 21% of Dau was released in pH 7.4 PBS, suggesting a property of pH-sensitive characteristic. To evaluate the target effect of the cRGD-linked micelles, cellular uptake of free Dau, PEG-PLA-Dau and cRGD-PEG-PLA-Dau micelles were analyzed by CLSM measurements. The endocytosis of Dau or micelles could be determined by the inherent fl uorescence of Dau. As shown in Figure 3(A), cRGD decoration on the surface of micelle signi fi cantly enhanced the cellular uptake by C6 cells after 1 h treatment. Compared with undecorated micelles group, more fl uorescence was detected in C6 cells incubated with RGD-decorated micelles, indicating a key role of RGD decoration in enhancing the cellular uptake. For 3 h treatment (Shown in Figure 3(B)), the fl uorescence of cRGD-PEG-PLA-Dau micelles was still higher than that undecorated micelles, but the ratio of fl uorescence intensity between RGD-decorated micelles and undecorated micelles dropped from 3.7 to 1.4. The decreasing ratio between two micelles may be attributed to the different uptake pathway of RGD-decorated micelles and undecorated micelles. As reported previously, nanopar- ticles are usually non-speci fi cally internalized into cells via endocytosis or phagocytosis; but the a v b 3 integrins could quickly pass through endosomes and reach the perinuclear compartments nearly 30 min after internalization (35). By cRGD modi fi cation the cRGD-PEG-PLA-Dau micelles could bind to the a v b 3 integrins expressed on the C6 cell surface and then endocytosed rapidly than undecorated micelles. As the incubation time prolonged to 3h, the supe- riority of these endocytosis was not obvious than undecorated micelles. The endocytosis rate was also measured by fl ow cytometry analysis. As shown in Figure 4, both PEG-PLA-Dau and cRGD-PEG-PLA-Dau micelles could be effectively uptake by C6 cells. As time prolonged to 3 h, more micelles were endocytosed. The cRGD-decorated micelles showed enhanced endocytosis by C6 cells as seen in CLSM images. Moreover, as the time prolonged to 3 h, the difference in cellular uptake between two micelles gradually decreased, which were consistent with the results in CLSM. The results indicated that RGD decoration play a key role in improving the tumor cell uptake of Dau- loaded micelles. The cytotoxicity of PEG-PLA-Dau and cRGD-PEG- PLA-Dau micelles were also examined in C6 cells by MTT assay. The C6 cells were incubated with PEG-PLA- Dau and cRGD-PEG-PLA-Dau micelles at various concentrations with free Dau as a positive control for 24 h and 48 h. The cell viability of 24 h and 48 h were shown in Figure 5 (A) and (B), respectively. All the micelles showed a time and concentration dependent manner of tumor cell proliferation inhibition effect. The results showed that free Dau displayed higher cytotoxicity than two micelles for 24 h and 48 h treatment, which could be attributed to the most cell uptake as much stronger fl uorescence was seen in the CLSM image. As for two micelles, cRGD decorated micelles displayed higher cytotoxicity at all concentrations than undecorated micelles. The higher cytotoxicity of RGD-decorated micelles could be explained by more cellular uptake of micelles as seen in CLSM. These results suggested a positive correlation between cellular uptake and cytotoxicity. In summary, we successfully developed cRGD decorated Dau-conjugated micelles for the treatment of C6 glioblastoma cells. The micelle displayed nearly sphere structure and had a diameter of about 50 nm. Enhanced uptake of cRGD decorated micelles by C6 cells were observed via CLSM and fl ow cytometry experiments. The cRGD decorated micelles also showed higher cytotoxicity to C6 cells than undecorated micelles. Our study demonstrated that cRGD functional micelles could be used to target delivery of anti-tumor drugs to glioblastoma. This work was fi nancially supported by National Key Technology R&D Program (No. 2012BAI29B05), National Natural Science Fund (No .81150015), Jilin Pro- vincial Scienti fi c and Technologic Development Project (No.20111809), Project of Technology Department of Jilin City (No.201233126 and 201233120) and Doctoral Science Foundation of Beihua University (5210235013081). The authors declare that there is no con fl ict of ...
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... cRAD-micelles), and provided the highest growth inhibitory effect (about 6-fold more compared to cRAD-micelles) [93]. A second study tested these micelles against lymph node metastasis in a syngeneic Guan and coworkers exploited cRGDfK in various micellar systems to achieve enhanced tumor targeting [95][96][97]. Integrin-targeted hybrid micelles of 18 to 60 nm were constructed in aqueous media by coassembling 20% cRGDfK-PEG-PLA and 80% epirubicin (EPI) conjugated to mPEG-poly(allyl glycidyl ether) (mPEG-PAGE-EPI). In this case, EPI was conjugated via pH sensitive carbamate or hydrazone linkers. ...
Targeting angiogenesis-related pathologies, which include tumorigenesis and metastatic processes, has become an attractive strategy for the development of efficient guided nanomedicines. In this respect, integrins are cell-adhesion molecules involved in angiogenesis signaling pathways and are overexpressed in many angiogenic processes. Therefore, they represent specific biomarkers not only to monitor disease progression but also to rationally design targeted nanomedicines. Arginine-glycine-aspartic (RGD) containing peptides that bind to specific integrins have been widely utilized to provide ligand-mediated targeting capabilities to small molecules, peptides, proteins, and antibodies, as well as to drug/imaging agent-containing nanomedicines, with the final aim of maximizing their therapeutic index. Within this review, we aim to cover recent and relevant examples of different integrin-assisted nanosystems including polymeric nanoconstructs, liposomes, and inorganic nanoparticles applied in drug/gene therapy as well as imaging and theranostics. We will also critically address the overall benefits of integrin-targeting.
Metal complexes have recently attracted a lot of attention, particularly as anticancer and antibacterial agents, owing to their versatile mechanisms and easily tunable characteristics. However, the application of such compounds in a medical context, especially in cancer treatment, has been hampered due to their limited solubility and stability in water and aqueous solutions, as well as a lack of selectivity. To address these limitations, several approaches have been developed to improve the targeted delivery of these compounds and their solubility. The first strategy involves the physical encapsulation of these metal complexes within carriers to facilitate controlled drug release. The second strategy involves covalently linking metal complexes to polymers, creating prodrugs that can be converted to active drugs at a more controllable rate. Despite the increasing variety of polymers available, the need to develop those able to be metabolized by the body, producing non‐toxic residues, remains crucial for effective treatment, particularly in the area of diseases such as cancer. In this perspective article, an overview of recent developments in the encapsulation of metal complexes using biodegradable polymers for cancer therapy is provided. It is specifically highlighting how the formed nanoparticles (NPs) enhance the selectivity and toxicity toward cancer cells compared to the parent metal complex, while simultaneously reducing the harmful side effects of traditional chemotherapies, opening the door for using them in combination therapies (PDT, PTT).
There are a large number of pharmaceutical products in the market containing heterocyclic compounds. Heterocyclic compounds are explored in the field of therapeutics due to their unique physicochemical and pharmacological properties. A large number of heterocyclic compounds existing in the pharmaceutical market have marked anticancer activity and many of them are under research investigations to treat different types of cancers. Anticancer heterocyclic compounds show many shortcomings such as other anticancer agents in bioavailability and site-specific drug delivery resulting in toxicity and decreased patient compliance. These shortcomings can be eliminated by applying the principles of nanotechnology. The present review discloses the biochemical mechanism of action, different biological targets, intrinsic shortcomings, and structure-activity relationships of anticancer heterocyclic compounds. Furthermore, the role of different nanocarrier systems in selective biological targeting and alteration of pharmacokinetic and pharmacodynamic characteristics of anticancer heterocyclic compounds will be discussed in detail.
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This work aims to study the construction of reverse aspirin-loaded micelles prepared from amphiphilic PEG-PLA-SA triblock copolymers and the optimization of the preparation process. Using polyethylene glycol (PEG) as the initiator, ring-opening polymerization of L-lactide (L-LA) was used to prepare PEG-PLA diblock copolymers. Final product PEG-PLA-SA triblock copolymers were prepared by the reaction of stearic acid (SA) and PEG-PLA catalyzed by 4-dimethylaminopyridine (DMAP) and N,N’-Dicyclohexylcarbodiimide (DCC). Fourier transform infrared spectrometer (FT-IR) was used to characterize the product structure. PEG-PLA-SA triblock copolymers self-assembled in toluene/ethanol/water system to form reverse micelles, which could encapsulate aspirin into a hydrophilic core. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) were used to determine the size and morphology of reverse micelles. The results showed that the reverse micelles are spherical, with a particle size of less than 70 nm. Response surface analysis method was applied to optimize the preparation process of PEG-PLA-SA. In vitro drug release was achieved by embedding reverse aspirin-loaded micelles in the biocompatible membrane in phosphate buffer saline (PBS) at 37°C. In the first 8 h, the drug release rate of the triblock copolymers was slower than that of the diblock copolymers. After 8 h, the drug release rate of both tended to be flat. The stability of aspirin-loaded reverse micelles was studied through accelerated test. These results indicate that reverse micelle PEG-PLA-SA may be a promising carrier for hydrophilic drugs like aspirin.
Nowadays development of safe and simple siRNA delivery system is still a great challenge. Carbon dots have attracted considerable attention in bioimaging, drug delivery, nanosensors, and other fields during the past few years. In this study, we successfully developed carbon dots (TEPA-CDs) as siRNA nanocarriers based on glucose and tetraethylene pentamine. TEPA-CDs have a mean diameter of less than 10 nm and positive charged decoration. TEPA-CDs could condense siRNA into stable complexes without no obvious premature release. Cellular uptake analysis clearly showed that Cy3-labeled siRNA could be uptake by HeLa cells. GFP expression in HeLa-EGFP cells could be significantly inhibited by TEPA-CDs/siRNA complexes. Our study indicated that TEPA-CDs could be used as a siRNA nanocarrier to tumor cells.
Although there is a large number of studies available evaluating the therapeutic efficacy of targeted polymeric nanoparticles, little is known about the critical attributes that can further influence their uptake into target cells. In this study, varying cRGD ligand densities (0-100% surface functionalization) were combined with different PEG spacer lengths (2/3.5/5kDa) and the specific receptor binding of targeted core-shell structured PLGA/PLA-PEG nanoparticles was evaluated using αvβ3 integrin overexpressing U87MG glioblastoma cells. Nanoparticles with 100% surface functionalization and short PEG2k linkers displayed a high propensity to form colloidal clusters, allowing for the cooperative binding to integrin receptors on the cellular membrane. In contrast, the high flexibility of longer PEG chains enhanced the chance of ligand entanglement and shrouding, decreasing the number of ligand-receptor binding events. As a result, the combination of short PEG2k linkers and a high cRGD surface modification synergistically increased the uptake of nanoparticles into target cells. Even though to date, the nanoparticles size and its degree of functionalization are considered to be major determinants for controlling the uptake efficiency of targeted colloids, these results strongly suggest that the role of the linker length should be carefully taken into consideration for the design of targeted drug delivery formulations in order to maximize the therapeutic efficacy and minimize adverse side effects.
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Poly(ethylene glycol)-poly(lactic acid) copolymer, prepared by ring opening polymerization, was used as a single platform to co-deliver both hydrophilic doxorubicin and hydrophobic docetaxel (DTX) in a simulated physiological environment. The average size of the negatively charged drug loaded polymeric micelles were found to be 293 nm. The drug loading (%) and encapsulation efficiency (%) were calculated to be 1.21 and 59.0, respectively. The in vitro cytotoxicity test using MCF7 breast cancer cells was conducted using 1 × 10⁴ cells in 10% FBS and 1% antibiotic, and the absorbance of formazan was evaluated at 570 nm. Cell growth inhibition by MTT assay showed viability of 33% of the MCF7 cells after treatment with drug-loaded micelles for 48 h. Controlled release of drugs from the polymeric micelles indicated a burst release effect initially; whereas, 98% of drug could be released at pH 7.4 within a time period of 96 h. Time period for drug release shorten to 48 h only in simulated mild acidic pH (5.4) condition. The in vitro drug release study from micelles indicated synergistic cytotoxicity effect in human metastatic breast cancer MCF7 cell.
