Preparation Of MPEG-PCL Polymersomes And Release Investigation Of Insulin-loaded MPEG₁₁₄-PCL₁₅₂ Polymersomes

Background and objectiveDue to the properties of rapid elimination and metabolism in vivo, poor tissue penetration, low bioavailability and other defects, the clinical use of therapeutic protein peptides are facing an enormous challenge. Insulin(INS) is a common drug for diabete patients, however, oral administration of insulin is difficult due to its low bioavailabilty, susceptible to enzymatic degradation in the digestive system. Insulin usually administrated via daily subcutaneous injection. Disadvantages of insulin daily subcutaneous administration include local pain, discomfort, infection, immune reaction, lipoatrophy and lipohypertrophy at the injection site. Therefore, design long-term and non invasive insulin delivery system has been one of the research hot spot worldwide. Using polyacticacid(PLA), polycaprolactone(PCL), poly(lactic-co-glycolicacid) (PLGA) and other polyester materials as the delivery carrier for insulin, although they can effectively protect the insulin, the drug loading amount is too low and the particles may be easily swallowed up and eliminated by the endoplasmic reticulum. With the emergence of amphiphilic block copolymer, it can effectively reduce the disadvantages of using single polyester material above. In recent years, the amphiphilic polymeric particle as an effective drug carrier transport has advantages of good drug loading capacity, good stability, long circulation time in vivo, being targeted modified, sustained release and targeting property, attracted widespread attention. The hydrophilic chain segment PEG is the most commonly hydrophilic molecules used to modified other hydrophilic materials and own many good characters such as excellent biocompatibility, strong hydrophilicity, high cell penetrability, non-toxic and non-immunogenic in vivo. And PCL as a most attractive medical polymer material with excellent properties such as biocompatibility, biodegradability, non-toxic, drug permeability, has been approved by FDA to use for human.Polymersomes(PS) were self-assembled by the amphiphilic block copolymer when they were dispersed in solution, which have the sphere structure like liposome with a hydrophilic inner cavity. The hydrophilic cavity can be used to encapsulate the hydrophilic drugs such as enzymes, DNA, RNA and protein peptides; then the lipid-soluble drugs can be encapsulated in the hydrophobic layer. Compared with other particles, polymersomes have a better ability to load the hydrophilic drugs because of its larger inner cavity’s volume. Moreover, the double membrane structure of polymersomes makes themselves more stable, and their hydrophilic membrane can effectively avoid the endoplasmic reticulum when administrated in vivo, with the effect of prolonging the circulation time and improving the ability of passive targeting. In consequence of the special components of mPEG-PCL copolymer, the polymersomes possess better physical and chemical properties:For instance,adopting the method of changing the ratio of the hydrophilic block to the hydrophobic block or the type of the copolymer can lead to a better property of the polymersomes; Futhermore,regulating the particle size of polymersimes is also accessible; Otherwise,the structure of two layer films for polyersomes can be utilized to load drags of different polarities; Finally, compared with liposomes and other small molecular surfactant vesicles, the structure of polymersomes has the features such as higher bonding strength,more stable performance and much higher drug-loading capacity.Considering the above research backgrounds, in this study, we firstly synthesized the amphiphilic block copolymer of different molecular weight, then investigated the particle size, the polydispersity index and the critical aggregation concentration(CAC), which may be influenced by different preparation methods and block ratio, in order to provide theoretical basis for seeking the best preparation condition. Insulin, used as the model drug to preparate the insulin-loaded mPEG-PCL polymersomes, can be loaded into the inner cavity during the hydration process because of the same polarity as the inner cavity. The polymersomes structure can not only protect the insuin from degradation to some extent in vivo, but also achieve a sustained release effect when the particles structure disintegrated.Methods1. Synthesis and characterization of different molecular weight mPEG-PCL copolymerThe different molecular weight mPEG-PCL copolymer were synthesized via ring-opening polymerization by using mPEG5k and ε-CL at a mass ration of 1:2,1:3, 1:4 with stannous octoate(1% mol ε-CL) as catalytic agent, respectively. Their structure were characterized by Fourier transform infrared spectroscopy (FT-IR) and proton nuclear magnetic resonance spectroscopy (1H-NMR). The molecular weight were also calculated according to NMR spectra.2. Preparation and characterization of different molecular weight mPEG-PCL polymersomesThe amphiphilic block polymersomes were prepared by film-hydration method and solvent evaporation method respectively. The particle size were examined by dynamic light scattering, as to select the appropriate polymersomes preparation method. Then the critical aggregation concentration (CAC) was detected with fluorescence techniques using pyrene as a probe.3. Preparation and characterization of insulin-loaded mPEG114-PCL152 polymersomesThe mPEG114-PCL152 amphiphilic block copolymer was chosen as the delivery carrier for insulin, prepared the insulin-loaded mPEG114-PCL152 polymersomes. The encapsulation efficiency and drug loading rate were determined by Bradford method. Effects of different dosing drug ratio to the encapsulation efficiency and drug loading rate were evaluated, selected the most suitable conditions for preparation.4. In vitro and in vivo release investigation of insulin loaded mPEG114-PCL152 polymersomesDrug loaded polymersomes solution was placed in phosphate buffer solution (PH=5.3) at 37℃ with gentle stirring. At predetermined time points, the samples were taken out, with 12000 r-min-1 25 minutes centrifugal treatment. The supernatant was taken for insulin content determination and calculate the cumulative release amount. Furthermore, the drug release mechanisms were studied with different mathematical models. The diabetic mice models were established by using streptozotocin (STZ), subcutaneous injected of drug loaded polymersomes. At predetermined time points, the mice blood glucose value were determined by the blood glucose meter, studied the effect of the drug loaded polymersomes to the hypoglycemic effect.Results1. Synthesis and characterization of different molecular weight mPEG-PCL copolymerThree kinds of different molecular weight amphiphilic block copolymer were successfully synthesized by using mPEG and ε-CL according to the results of FT-IR and’H-NMR. Their molecular weight were 14665,19130 and 22350, calculated according to NMR spectra, whose structure type were respectively mPEG114-PCL85, mPEG114-PCL124 and mPEG114-PCL152。2. Preparation and characterization of different molecular weight mPEG-PCL polymersomesThe particle size were (204.0±30)nm, (202.8±5.2) nm, (182.4±9.7)nm, the polydispersity index (PDI) were 0.390,0.306,0.202 and the critical aggregation concentration(CAC) were 5.12×104 mg·mL-1,5.01×10-4 mg·mL-1 and 4.07×10-4 mg·mL-1 respectively, which were prepared by film hydration method. Compared with the solvent evaporation method, the former method prepared particles were more uniform and stable, especially the mPEG114-PCL152 polymersomes particles.3. Preparation and characterization of insulin loaded mPEG114-PCL152 polymersomesThe drug encapsulation efficiency were (42.90±0.57)%, (62.80±2.14)%, (33.75±1.62)% and the drug loading rate were (4.11±0.05)%, (11.10±0.34) %, (11.90±0.50)% according to the 10% wt,20% wt,40% wt dosing drug ratio. It is visible to find that when prepared at 20% wt dosing drug ratio we can get the highest utilization rate of the model drug.4. In vitro and in vivo release of insulin loaded mPEG114-PCL152 polymersomesThe drug loaded polymersomes were with busrt release at initial 2 hours, then within 48 hours the accumulated release amount was about 55.05%. From the release curves, we can find that the polymersomes showed a good sustained-release without the initial 2 hours. The release model was much more closed to the Higuchi model. Then in vivo release study, it showed a good sustained-release, which can effectively reduce the blood glucose value for a long time.ConclusionWe successfully synthesized the amphiphilic block copolymer mPEG-PCL with using mPEG as the hydrophilic block and PCL as the hydrophobic block. The particle size were small and uniform distribution. Taking mPEG114-PCL152 as the delivery carrier for insulin, and the drug loaded polymersomes showed a good sustained-release effect in vivo and in vitro study.