Biodegradable Crosslinked Nanomedicines For Efficient Tumor Targeting Chemotherapy And Diagnosis

The morbidity and mortality of cancer increased dramatically in recent years. Development of efficient and safe therapeutics is one of the most important missions for researchers. Nanomedicines, offering improved drug solubility, prolonged circulation time in vivo, increased drug accumulation in tumor sites via EPR effect, have showed tremendous potential. Among them, Doxil?, pegylated lipsomal doxorubicin(DOX), is the first antitumor nanomedicine approved by FDA to treat Kaposi’s sarcoma, ovarian cancer and multiple myeloma. Abraxane?, a paclitaxel loaded albumin nanoparticles, is approved to treat metastatic breast cancer, pancreatic cancer and non-small cell lung cancer. The approval of the first polymeric nanomedicine, GenexolTM(Samyang Corporation, South Korea) based on paclitaxel loaded PEG-PLGA nanoparticles motivated researchers to translate a series of polymeric nanomedicines into clinical trials of different stages including NK911, NK105, and NK102. The success of polymeric nanomedicines inspire scientists to explore more multi-functional nanocarriers for tumor targeted treatment. Polymers based on polyesters, polypeptides and polycarbonates attract wide attention due to their superior biocompatibility,biodegradability and nontoxicity of degradation product. However, these nanomedicines encountered four obstacles in translation into clinics which need to be overcome, namely, insufficient stability in vivo, poor tumor tissue penetration, low tumor cell uptake and slow drug release intracellularly. Development of highly efficient, safe and nontoxic polymeric gene vectors as well as multi-functional platform for theranosis is of demand. In chapter 1, we present a brief literature overview on the current status, challenges or obstacles and solutions of tumor therapy.To this end, in this thesis we have designed a multifunctional platform for treating cancers, based on a series of tumor active targeting, reduction sensitive, self crosslinked biodegradable polymeric micelles and polymersomes. We found that these nanomedicines have outstanding performance in various tumor models in mice. In chapter 2, we designed novel dithiolane trimethylene carbonate(DTC), and obtained biodegradable amphiphilic copolymers polyethylene glycol-b-poly(dithiolane trimethylene carbonate)(PEG-b-PDTC) and c RGD decorated PEG-b-PDTC. Via their co-self-assembly DOX loaded c RGD decorated, self-crosslinked, reduction-sensitive micelles(c RGD/DOX-SCID-Ms) were prepared for active targeting to αvβ3 over-expressing neovasculature and maligant tumor cells like B16 melanoma cells. c RGD/DOX-SCID-Ms displayed apparent reduction sensitivity by showing great stability in physiological environment while quick de-crosslink and drug release in an intracellular mimicking conditions containing 2~10 m M glutathione. Moreover, they showed superior in vivo performance that c RGD/DOX-SCID-Ms had elimination half-time t1/2,β of 6.19 h(0.18 h for free DOX), DOX accumulation in tumor site of 6.13%ID/g at 6 h post iv injection. What is more extraordinary is that c RGD/DOX-SCID-Ms had maximum tolerant dosage(MTD) of >100 mg/kg(>20-fold higher than that of free DOX), high antitumor activity and low side-effects in B16 tumor bearing mice. The results revealed the superiority of the present nanosystem.The success in DTC containing micellar nanomedicines encourages us to prepare self-crosslinked polymersome nanomedicines in chapter 3. Here with based on DTC and trimethylene carbonate(TMC) we have devised and synthesized amphiphilic copolymers PEG-P(TMC-DTC) and c RGD-PEG-P(TMC-DTC). By regulating the molecular weight ratio between hydrophilic and hydrophobic blocks polymersomes could be obtained. Via co-self-assembly, p H gradient active loading of doxorubicin hydrochloride(DOX·HCl) and self-crosslinking, tumor targeting robust polymersome nanomedicine c RGD-PS-Dox were prepared. c RGD-PS-Dox with drug loading content of 15.4 wt.% was of 120 nm(PDI 0.1), and selectively uptaken by αvβ3 integrin over-expressing U87 MG cells via receptor-mediated endocytosis showing high cytotoxicity(IC50 of 1.77 μg/m L). Moreover, in mice c RGD-PS-Dox exhibited prolonged circulation time(t1/2,β of 4.49 h vs. 0.18 h for DOX·HCl), quickly enhanced drug accumulation in tumors(6.78%ID/g), superb antitumor activity and low systemic toxicity. Thus, c RGD-PS-Dox expends the applications of DTC based nanomedicines for tumor targeted drug delivery and the result serves as solid base for investigation in other tumor models.Lung cancer with a high mortality and increasing morbidity has become one of the most lethal malignancies worldwide. Chapter 4 presents three lung cancer targeting polymersome nanomedicines, c RGD-PS-Dox, c NGQ-PS-Dox and c NGQ-PS-DTX, which were evaluated in nude mice bearing subcutaneous A549(non small lung cancer cell) tumor model(sub-A549) and orthotopic tumor model(orth-A549). Three nanomedicines all displayed high tumor accumulation, powerful tumor inhibition, prolonged survival time and low toxicity in sub-A549 model. To our greatest surprise, in orth-A549 model the targeted therapy effect is more prominent. Especially, c RGD-PS-Dox and c NGQ-PS-Dox could effectively suppress tumor growth,metastasis and shrank the tumors after 16 d treatment without toxicity, 100% mice survived the 45 d experiment period. Interestingly, the commercial pegylated liposomal DOX(Lipo-Dox) restrained tumor growth but with concurrent damage to normal tissues and all mice died in 32 d. Therefore, the polymersome system apply for both hydrophilic and hydrophobic drugs,for different targeting ligands, all could hold back tumor growth and metastasis of lung cancer. Thus the polymersome nanomedicine platform have made a giant leap for clinical translation.The exciting results as well as the structural similarity between polymersomes and liposomes drive us in chapter 5 to directly compare our polymersome nanomedicines with Lipo-Dox in treating malignant ovarian cancer, which in clinic uses Lipo-Dox as first-line treatment. We prepared GE11-PS-Dox, in which GE11 has high affinity to epidermal growth factor receptor(EGFR) over-expressing SKOV-3 ovarian cancer cells. GE11-PS-Dox has MTD of 160 mg/kg, 8-fold higher than Lipo-Dox, thus significantly increases the therapeutic widow of DOX. Excitingly, GE11-PS-Dox at DOX dosage of 60 mg/kg(single injection) offered better treatment benefit than Lipo-Dox with effectively inhibited tumor growth, extended survival time and lowered side effects. Thus GE11-PS-Dox opens the possibility as an better alternative to Lipo-Dox in ovarian cancer therapy.To cope with the low tumor penetration of nanomedicine, in chapter 6 we have devised i RGD peptide coupled PS-Dox(i RGD-PS-Dox), in which i RGD has strong tumor permeability, high affinity to neovascular as well as αvβ3 over-expressing B16 melanoma. The effect of surface density of i RGD on blood circulation, tumor accumulation, tumor penetration and tumor inhibition has been systematically investigated in B16 bearing C57BL/6 mice. It was illustrated that 50% i RGD had the longest circulation time(4.19 h), the highest DOX tumor accumulation(4.83%ID/g), the best antitumor efficacy and longest survival time. These results revealed the importance of tumor penetration in enhancing the efficacy of chemotherapy of polymersome nanomedicines in vivo.Biopharmaceutics like nucleic acids and proteins, showing high antitumor efficacy and selectivity with low toxicity, are superior over chemotherapeutics, especially si RNA which has direct antitumor effect. In chapter 7, we have extended the application of our polymersome nanomedicine to si RNA delivery into A549 cells in vitro and in vivo. We conjugated 1.8 k branched polyethylene imide(PEI) to the polymer end yielding PEG-P(TMC-DTC)-PEI, and via co-self-assembly and electrostatic interaction, si RNA was complexed with PEI located in the polymersome inner shell, offering si RNA-c NGQ/RCCPs. PEI is used here not only for loading si RNA but also for endosomal escape to realize the high efficient si RNA delivery in vivo. The studies displayed that si RNA-c NGQ/RCCPs had a prolonged circulation time(t1/2,β = 1.78 h vs. 0.12 h for naked si RNA), high si RNA accumulation in the lung site in orth-A549(4.03%ID/g). What’s more, therapeutic gene si PLK1 loaded c NGQ/RCCPs could suppress the tumor growth and metastasis, and significantly prolong the medium survival time(54.0 d) than controls. The c NGQ/RCCPs presents as a multifunctional platform for highly efficient and nontoxic biotherapy, especially tumor targeted gene therapy.The lack of safe and accurate early diagnosis accounts for one main reason for the high cancer mortality. Noninvasive medical imaging diagnostic methods including CT are commonly used in clinics, however, large amount of contrast agents have to be used. In chapter 8, we have designed biodegradable nano-sized iodinated polymersomes as CT contrast agent for tumor diagnosis and theranosis. Firstly, we invented novel diiodinated trimethylene carbonate(IC) monomer and synthesized amphiphilic copolymer PEG-b-PIC and PEG-b-P(IC-DTC) of which iodine content was as high as 60.4 wt.%. Polymersomes based on PEG-b-PIC(IPs) showed significantly better contrast efficiency than small molecular contrast agent iohexol at the same iodine content in vitro and in vivo. Moreover, IPS had a long circulation time(t1/2,β = 3.73 h), could enhance CT value of 43.2 Hu in the subcutaneous tumor in U87 MG bearing mice and of 121.3 Hu in the lung site in orthotopic A549 tumor bearing mice, which establish solid foundation for tumor early diagnosis. In the following, we applied c RGD directed, self-crosslinked polymersome nano contrast agent, c RGD-RCIPs-Dox, for tumor targeted imaging and therapy in B16 bearing mice. To our satisfactory, c RGD-RCIPs-Dox showed tumor specific contrast and 4 h post i.v injection, the CT enhancement at tumor site was 16.4 fold high as that of iohexol. Importantly, c RGD-RCIPs-Dox demonstrated a prolonged circulation time(t1/2,β = 6.84 h), a high DOX tumor accumulation(6.68%ID/g). These results explain its specific tumor imaging, enhanced anticancer effect and low side-effects. Thus c RGD-RCIPs-Dox combines advantegeous features like active tumor targeting, high loading of anticancer drug, reduction triggered intracellular drug release, high therapeutic efficiency as well as specific tumor imaging, and is very appealing in potential applications in tumor early diagnosis and targeted cancer theranosis.At last, chapter 9 overviews the whole thesis and gives a future perspective in this field.