Construction And Antitumor Effect Study Of Drug-Loaded Polymersomes Incorporated With Functional Small Molecules

Health is an eternal pursuit of mankind.In the field of cancer therapy,various nano-drug delivery systems have received tremendous attention in recent decades,among which the polymersome,also termed polymeric vesicle,is particularly eye-catching.Taking advantage of higher molecular weight and more functional modification possibilities,polymersomes exhibit enhanced stability as well as superior versatility compared to their lipid counterparts.At the same time,compared with micelles,polymersomes can simultaneously load hydrophobic and hydrophilic agents owing to the unique microstructure in which a watery interior is surrounded by hydrophobic membrane.Above advantages make polymersome distinguished from many other nanoparticles and gained extensive concern.However,polymersome-based nanomedicines for cancer therapy still face challenges.In particular,when delivering water-soluble chemotherapeutics at a high loading content,polymersomes usually suffer from premature drug leakage during blood circulation,which may result in severe side effects and ultimately undesirable therapeutic efficacy.On the other hand,the development of multidrug resistance is a major cause of clinical chemotherapy failure.Therefore,improving the delivery stability of polymersomes by inhibiting premature drug leakage,and endowing them with drug resistance-reversal ability will be crucial for the development of polymersomes in the field of cancer therapy.In this study,polymersomes constructed from a novel kind of amphiphilic polyphosphazene PEBP was designed and applied for the effective delivery of water-soluble chemotherapeutics.In order to address challenges encountered by polymersomes including premature drug leakage and drug resistance during cancer treatment,a series of work has been carried out and the main contents as well as conclusions are summarized below:（1）Firstly,amphiphilic grafted poly[（PEG）_x（BIMA）_yphosphazenes]_n（PEBPs）using（1H-benzo-[d]imidazol-2-yl）methanamine（BIMA）as hydrophobic side groups and poly（ethylene glycol）（PEG）as hydrophilic groups at different hydrophilic/hydrophobic ratios were designed and synthesized.The structures of obtained polymers were characterized by gel permeation chromatography（GPC）,nuclear magnetic resonance（¹H NMR）,and fourier infrared spectroscopy（FT-IR）.Self-assembly behaviors and doxorubicin hydrochloride（DOX·HCl）loading capacity of PEBPs were investigated using a simple dialysis method.Then PEBP-4 that can self-assemble into polymersomes was selected for further study.The encapsulation efficiency（EE）and loading content（LC）of DOX·HCl reached 98.23%and 12.83%,respectively.In vitro drug release study revealed that though the drug-loaded PEBP polymersomes exhibited relatively sustained release characteristics compared with free DOX·HCl,the accumulative release ratio under physiological conditions（p H 7.4）was still as high as 31%and 59%at 2 h and 24h,respectively,indicating that premature drug leakage would arise during blood circulation.（2）With the aim of enhancing packing density of polymersome’s hydrophobic membrane so as to inhibit burst release of DOX·HCl,a rigid cholesteric molecule,cholesteryl hemisuccinate（Chol HS）,was introduced into PEBP polymersome’s hydrophobic membrane by virtue of noncovalent interactions between Chol HS and benzimidazole group in the hydrophobic domain of PEBP.Self-assembly results of Chol HS and PEBP showed that when Chol HS content ranged from 1 wt%to 15 wt%,the system was clear and uniform,while phase separation occurred once Chol HS amount exceeded 15 wt%.Investigations including transmission electron microscopy（TEM）observation,dynamic light scattering（DLS）measurement,and drug loading capacity evaluation revealed that the incorporation of Chol HS（1-15 wt%）had negligible effect on the morphologies,particle sizes,and high DOX·HCl loading capacity of PEBP vesicles.Encouragingly,further in vitro release study indicated that Chol HS could effectively inhibit DOX·HCl release from PEBP polymersomes at p H 7.4,and the higher the content of Chol HS,the more effective the inhibitory effect was.For example,when Chol HS increased to 15 wt%,the release of DOX·HCl at 2 h and 24 h under p H 7.4 decreased to13%and 29%,respectively.Meanwhile,the inherent acid-triggered rapid drug release of PEBP polymersomes was not affected by Chol HS.In addition,cholesterol and cholesterol acetate were also introduced to PEBP polymersomes for comparison.As a result,the maximum co-assembled amount of either cholesterol or cholesterol acetate was only 1wt%,and their inhibitory effects on premature drug leakage is far weaker from that of 15wt%Chol HS.The superior compatibility between Chol HS and PEBP was confirmed by differential scanning calorimetry（DSC）and X-ray diffraction（XRD）.Furthermore,2D¹H-¹H NOESY NMR spectra,FT-IR analyses were used to investigate the underlying mechanisms,among which hydrogen bonding played a key role.Cytotoxicity study indicated that the IC₅₀ of PEBP-D-Ps and PEBP-Chol HS-D-Ps against S180 cells after24 h were 1.804μg/m L and 1.778μg/m L,respectively,showing comparable cytotoxic efficacy with that of free DOX·HCl(IC₅₀=1.737μg/m L).In vivo pharmacokinetic study confirmed the extended blood circulation of DOX·HCl-loaded polymersomes after Chol HS incorporation.For PEBP-Chol HS-D-Ps,the half-life(t_1/2β)was 4.29-fold longer than that of PEBP-D-Ps,and 8.55-fold longer than free DOX·HCl.Finally,the in vivo antitumor effects of various formulations were explored using mice bearing subcutaneous S180 tumors.It is inspiring that the tumor growth was remarkably suppressed by PEBP-Chol HS-D-Ps with a tumor inhibition rate of 73.56%（16 days）,and the cardiotoxicity of DOX·HCl was evaded.In summary,incorporation of Chol HS into PEBP polymersomes significantly improved the delivery stability of hydrophilic chemotherapeutics,ensuring the effectiveness as well as safety of cancer chemotherapy.（3）Aiming at endowing the nanovesicle system with tumor drug resistance-reversal capacity in addition to inhibiting premature drug leakage,vitamin E succinate（VES）,a vitamin E derivative,was proposed to introduce into the hydrophobic membrane of PEBP polymersome.VES is reported to target mitochondria and trigger a series of mitochondrial functional damage,induce tumor cell apoptosis,and reverse the resistance of cancer cells to chemotherapeutics by cutting of the intracellular energy source.It is worth mentioning that VES might also exhibit superior compatibility with PEBP owing to the existence of aromatic ring,carboxyl group and hydrophobic alkyl chain in its structure,which contribute to the multiple non-covalent interactions includingπ-πstacking,hydrogen bonding and hydrophobic interactions between VES and PEBP.First of all,PEBP bearing a slightly increased hydrophobic ratio compared with PEBP-4 in the previous chapter was synthesized with the expectation of improving the incorporated VES content.The self-assembled morphology of newly prepared PEBP was also typical vesicles under TEM observation,and high loading of DOX·HCl was achieved with the EE of 91.0%and LC of 9.2%.Moreover,PEBP polymersome exhibited superior VES loading capacity owing to the excellent compatibility,co-assembly feeding ratio of VES to PEBP could reach up to 40 wt%with the EE of 90.0%and LC of 26.2%,respectively.In vitro drug release study showed that VES significantly inhibited DOX·HCl release from PEBP polymersomes at p H 7.4,and the higher the VES content,the more effective the inhibiting.Especially when VES feeding ratio increased up to 40 wt%,the release of DOX·HCl at 24 h under p H 7.4 decreased from 41%to 13%,and the acid-accelerated drug release property remained.Compatibility investigation verified the existence of strong interactions represented by hydrogen bonding between VES and the hydrophobic side groups of PEBP.In addition,the biosafety of PEBP/VES system was confirmed by hemolysis test.K562/ADR cells with the resistance index（RI）against DOX·HCl of 158-fold and MCF-7/ADR cells with the corresponding RI of 798-fold was chosen for further investigation.In vitro cytotoxicity study showed that the incorporation of VES could significantly reduce the IC₅₀ of DOX·HCl on both cells.Combining the in vitro drug release and cytotoxicity results,PEBP-VES-D-Ps with the feeing mass ratio of VES to PEBP at 40 wt%,in which the actual encapsulating amount of DOX·HCl and VES was about 1:3,was screened out as the optimal preparation.Subsequent experiments showed that the addition of VES significantly increased the uptake of DOX·HCl by both K562/ADR and MCF-7/ADR cells,and promoted cell apoptosis.The results of mitochondrial-dependent pathway investigation showed that VES could stimulate excessive production of reactive oxygen species（ROS）within tumor cells,result in loss of mitochondrial membrane potential（ΔΨ_m）and reduce intracellular ATP level.Finally,K562/ADR tumors-bearing nude mice models were constructed and in vivo antitumor study was carried out.The tumor inhibition rate of PEBP-VES-D-Ps was as high as 82.38%（16 days）,and the cardiotoxicity of DOX·HCl was significantly reduced without damaging other major organs of mice.Overall,the introduction of VES not only improved the delivery stability of nanovesicles under physiological conditions,but also produced synergistic effect with DOX·HCl by virtue of its mitochondrial-targeting capacity,which ultimately realized robust reversal of tumor drug resistance.In summary,a novel p H-responsive nanovesicle self-assembled from amphiphilic polyphophzene-based PEBP was constructed and realized high loading of water-soluble chemotherapeutic DOX·HCl.In order to tackle issues including drug delivery stability deficiency and tumor resistance,functional small molecules,Chol HS and VES,were introduced into PEBP polymersomes membrane by virtue of noncovalent interactions,respectively.Investigations showed that Chol HS could effectively enhance vesicular membrane compactness and inhibited DOX·HCl leakage during blood circulation,thus improving the antitumor efficacy as well as reducing side effects.Furthermore,the incorporation of mitochondrial-targeted VES could not only inhibit burst release of DOX·HCl,but also exhibited synergistic effect with DOX·HCl to reverse drug resistance of tumors.In summary,a safe,simple and effective cancer therapy strategy based on nanovesicle system was provided.