| Because of the poor solubility, serious side effects, as well as multidrug resistance, the effectiveness of chemotherapeutic drugs is greatly limited in the clinical treatment of cancer. However, the development of new drug delivery systems provides a new way of thinking in order to enhance the anti-tumor effects of the drugs.Nanotechnology has increasingly occupied an important position in pharmaceutical field. Among them, the amphiphilic polymeric micelles which are likely to self-assemble into stable nanoparticles in aqueous solution have achieved substantial progress in recent years. With the possibilities of improved targeting of tumor tissues, increased intracellular drug concentration, control-released payloads, and eventually reversed MDR of cancer, amphiphilic polymeric micelles can be used as a vector for hydrophobic drugs, genes, metal drugs, etc. Among various strategies, a common approach is to decorate the micelles outer layer with specific ligands that can recognize the antigen or receptor on the target cell membrane to achieve active targeting. The unique three-dimensional architecture of cyclodextrins (CDs) with hydrophobic inner cavity endows them with the attracting character to form host-guest inclusion via hydrophobic and van der Waals effect, or other specific interactions. Taking advantage of this point, CDs have been widely used as drug carriers to enhance the solubility, stability, and bioavailability of hydrophobic drugs. Through the modification of hydroxyl groups in CD and the introduction of different substituent groups, numerous CD derivatives with different properties and functions are obtained.To achieve polymeric carriers with enhanced loading capacity of hydrophobic drugs and stronger capability to reverse MDR, herein, a series of copolymers (PELA-CD or PECL-CD) were designed by integrating β-CD and PEGylated poly(D,L-lactide) or PEGylated poly(ε-caprolactone). The introduction of folate moieties aimed to achieve active targeting in cancer treatment, which would be regarded as a model for the synthesis of other PELA-CD derivates with various targeting sides responsive to specific receptors on cancer cells. The study emphasized on the action mechanism of PELA-CD and PECL-CD to sensitize P-gp overexpressed MCF-7/ADR cells provided theoretical basis and experimental evidence for this polymeric micelle vector system to reverse P-gp mediated drug resistence and improve the effect of antineoplastic agents.The main content and conclusions are summarized below:Firstly, a series of amphiphilic copolymers were synthesized including PEG-conjugated CD (PEG5000-CD), PELA54, and PELA-CD or PECL-CD compounds with varied weight ratios of PEG to LA or CL. The copolymer named as PELA54-CD contains PELA blocks with a feed ratio of mPEG5000 to LA as 5/4 (w/w). The structures of polymeric compounds were confirmed by 1H NMR. Once hydrophobic PL A or PCL segments were introduced, the CMCs of the polymeric aggregates significantly decreased to 0.0016-0.01 g/L compared with that of PEG5000-CD, and the values gradually decreased with the increase of hydrophobic chain content. Aggregates containing the largest amount of PLA or PCL, PELA54-CD or PECL54-CD displayed the lowest CMC value in their series. The particle size of PELA-CD or PECL-CD was between 100-200 nm.Secondly, using the oil/water emulsion method, the DOX loading content (LC) of PELA54-CD and PECL54-CD micelles reached to 17.6% and 20.3%(DOX entrapment efficiency was above 75%), respectively, which was superior to P-CD (3.4%), PEG5000-CD (8.9%), and PELA54 (12.3%). The enhanced loading of DOX was attributed to the cooperation of the β-CD inclusion effect and the hydrophobic interactions of PLA or PCL with DOX. The particle sizes of the resultant DOX-loaded micelles were distributed between 100 and 160 nm, which was small enough to be transported from blood circulation to the tumor site via EPR effect and further taken up by tumor cells after intravenous injection. The release of DOX from PELA-CD/DOX or PECL-CD/DOX was accelerated under acidic conditions (pH5.5), but comparatively slow under physiological conditions (pH 7.4). The typical drug release pattern is conducive for the nanoparticles to play a cytotoxic effect in the tumor.Using docetaxel as another model drug, the method for determine DTX concentration via HPLC was established with high precision and conformed to the system suitability. By examining effects of different methods on characterization of DTX loaded PELA54-CD micelles, film hydration method was considered as the optimum way to achieve higher encapsulation efficiency and spherical shape, the particle size of DTX-loaded micelles were 80 to 120 nm. Similarly, PELA54-CD achieved the highest loading content and entrapment efficiency.Then a new synthetic route was developed to conjugate folate into PELA-CD to achieve active targeting system (FA-PELA-CD). FA-PELA-CD could self-assemble in aqueous solution and effectively entrap DOX molecules due to synchronized hydrophobic interactions between the drug and CD or PLA. The FA conjugation did not change the release pattern as PELA-CD, the release of DOX increased at pH 5.5. Therefore, once the micelle particles were endocytosed into the acidic endosomes or lysosomes of solid tumors (pH 5.5-6.0), DOX release was accelerated. This pH-dependent release property played a significant role in enhancing DOX action inside cancer cells.The in vivo antitumor effect of DOX-loaded FA-PELA-CD micelles was evaluated in a KB cell-xenografted nude mouse model. In the in vivo experiments of this part, PELA-CD/DOX and FA-PELA-CD/DOX induced a significantly higher survival rate than 5 or 10 mg/kg of free DOX·HCl, and markedly attenuated DOX-induced chronic damages. Compared to controls, treatment with 10 mg/kg FA-PELA-CD/DOX micelles induced a significantly greater tumor growth inhibition (86%) than treatment with the same dose of PELA-CD/DOX micelles (73%). Ex situ fluorescence imaging experiments demonstrated that FA-PELA-CD/DOX micelles induced an increased accumulation of DOX at the tumor site, interestingly,10 days after administration, tumors displayed a maximal DOX retention compared to other organs. The subsequent prolonged circulation half-life increased the probability of the PEGylated micelles to reach the tumor via EPR effects, which led to the enhanced efficacy of the treatment and alleviated toxicity. Moreover, histological examination further demonstrated irreversible and time-dependent damages in FA-PELA-CD/DOX treated tumor cells, and this formulation could significantly attenuate DOX-induced severe myocardial damages in heart due to the active targeting of folate conjugation.Lastly, the action mechanism of PELA54-CD micelle-mediated tumor suppression was validated using MCF-7/ADR cells and the zebrafish xenograft model. The experiements confirmed that PELA-CD and PECL-CD micelles could better enhance cellular uptake of R123, as well as improve the apoptosis and cytotoxicity of DOX against MCF-7/ADR cells than DOX·HCl solution, PELA, PEG-CD, or β-CD. The images and data in zebrafish assays showed that PELA54-CD helped to improve the retention of Rhodamine B in zebra fish in a concentration-dependent manner, which was consistent with the result of intracellular accumulation of R123 in MCF-7/ADR cells. Zebrafish xenografts were generated by microinjection of MCF-7/ADR cells labeled with CM-Dil into the yolk sac of embryos that were 48 h post-fertilization. The xenografts of MCF-7/ADR cells treated with PELA54-CD/DOX had weaker fluorescence intensity than that in the control group, indicating that PELA54-CD/DOX could kill MCF-7/ADR cells in zebrafish xenografts. The statistical results showed that, compared with the control group, the fluorescence intensity of tumor xenografts was reduced with increased DOX dosage after treatment with PELA54-CD/DOX containing 10,15, or 20 ng DOX. The corresponding inhibition rates of tumor growth reached 28±1.91%,47±2.66%, and 64±2.66%, for 10,15, and 20 ng, respectively. Meanwhile, compared with treatment with free DOX-HCl, the cardiac toxicity of DOX was decreased following PELA54-CD/DOX treatment. Therefore, PELA54-CD/DOX achieved a significantly higher anti-tumor efficacy than the same dose of free DOX-HCl and markedly attenuated DOX-induced chronic damages.This study also aimed to explore the action mechanism of PELA-CD and PECL-CD to reverse DOX resistance and clarify the relationship between the polymers’ action effect and their chemical structure. The assay of ATP depletion, ATPase activity, P-gp expression, lactate dehydrogenase, and mitochondrial membrane potential performed in MCF-7/ADR cells and the zebrafish xenograft model validated that the active mechanism PELA54-CD micelle-mediated tumor suppression was as follows:(1) induce intracellular depletion of ATP, (2) increase P-gp ATPase activity as a competitive substrate, and (3) decrease P-gp expression.In summary, this study constructed a new drug delivery system of amphiphilic polymeric micelles based on β-CD to improve the entrapment capability of hydrophobic drugs and reverse P-gp induced drug resistance. The modified tumor delivery system with FA enhanced toxicity of DOX, improved the tumor targeting ability and attenuated drug-induced side effects. A series study explored the active mechanism of PELA-CD to reverse DOX resistance and clarified the relationship between the polymers’ action effect and their chemical structure. The establishment of evaluation system for active mechanism of PELA-CD provides a platform for study of P-gp related drug resistance. |
PDF Full Text Request