Polymeric micro- and nano-scale systems as a platform for encapsulation and delivery of drugs based on resorbable polyhydroxyalkanoates
A drug delivery system (DDS) is defined as a formulation or a device that enables the introduction of a therapeutic substance in the body and improves its efficacy and safety by controlling the rate, time, and place of release of drugs in the body. Such systems are especially actual for treatment oncological and long current infectious diseases. Perspective material for creation such system are polyhydroxyalkanoates (PHAs). These polymers are biocompatible and inert towards animal tissues; in biological environments they are degraded to end products (CO2 and H2O). Investigations performed at the Institute of Biophysics, Siberian Branch, Russian Academy of Sciences and Siberian Federal University, revealed the high biocompatibility of high purity PHA samples at cellular and tissue levels, including contact with blood, as well as their applicability for the design of endoprostheses of various kinds, as matrices of functioning cells, and for deposition of drugs (Shishatskaya et al., 2008; 2011)
Work carried out in the framework of the mega-project, implemented under the supervision of leading scientists MIT professor "Biotechnology of new biomaterials" (USA) Anthony John Sinskey (RF Government Resolution № 220 of April 9, 2010), as a result of which it was possible to carry out comprehensive studies, including the development and optimization of methods for manufacturing micro- and nanoparticles from PHA by emulsion and spray-drying methods; research and identification of key factors influencing to the performance of engineered particles. Studying of the possibility of loading the wide range of drugs (cytotoxic, antibacterial and anti-inflammatory drugs) and the drug release in vitro. Research biocompatibility and adhesive properties of PHA microparticles with the assessment of the drug effectiveness of developed microcarriers.
Preparation and characterization of PHAs micro- and nanoparticles
The effect of the preparation technique (chemical composition of a polymer, type and method of emulsion mixing, and molecular mass of a drug) on the yield, structure, and size of microparticles obtained from resorbable polyesters of microbiological origin, polyhydroxyalkanoates, is studied. Microparticles made from degradable polyhydroxyalkanoates of different chemical compositions a homopolymer of 3-hydroxybutyric acid, copolymers of 3-hydroxybutyric and 4-hydroxybutyric acids (P3HB/4HB), 3-hydroxybutyric and 3-hydroxyvaleric acids (P3HB/3HV), 3-hydroxybutyric and 3-hydroxyhexanoic acids (P3HB/3HHx) were prepared using the solvent evaporation technique, from double emulsions. The present study revealed a significant effect of the chemical composition of the polymer on the average diameter and ξ-potential of microparticles. It is found that the concentration of the polymer solution and the method of emulsion mixing are the most significant factors affecting the diameter of microparticles based on polyhydroxyalkanoates; the surface structure of particles depends to a higher extent on the chemical composition of the polymer. The family of microparticles from 100–200 nm to 50–70 µm in diameter is synthesized. This study showed that the values of ξ-potential of microparticles prepared from different types of PHAs varied significantly. The lowest values of ξ-potential were recorded for PHB3/HHx microparticles (-32.2 mV) and the ξ-potential of P3HB was no higher than -11 mV. Also, the possibility of using the spray drying method for the construction of PHA based microparticles was studied. The dependence of characteristics of microparticles (yield, average diameter, zeta-potential) from processing parameters (temperature at the inlet of the system, the feed rate of polymer solution and concentration of the polymer solution) was found. It is shown that zeta potential of microparticles was influenced by concentration of the polymer solution, and the average diameter was determined by temperature of the inlet. The zeta-potential of microparticles prepared by spray drying method was lower ( from -66 to -115 mV ) than that of P3HB particles. The conditions of obtaining P3HB microparticles were optimized to minimize polymer loss.
It were prepared microparticles loaded with different drugs. The study investigates properties of microparticles prepared from PHAs depending upon the method of preparation employed and taking into account the size of particles maintained in liquid media.
It is shown that the rate of drug release from microparticles in vitro into the medium is higher in the case of 3-hydroxybutyrate copolymers with 3-hydroxyvalerate than in the case of the homopolymer of 3-hydroxybutyrate. This parameter increases with the content of 3-hydroxyvalerate units in the copolymer and the porosity and mass fraction of the drug in particles with a decrease in their sizes. For in vitro systems containing a phosphate buffer, variation in the preparation parameters makes it possible to obtain microparticles with various characteristics suitable for deposition of drugs. For microparticles obtained from polyhydroxyalkanoates and having different diameters, the mathematical description of the kinetics of drug release from the polymer matrix is provided. The value of ζ-potential was influenced by drug loading into microparticles; the drug loaded microparticles maintained in balanced phosphate buffer for 30 days had higher physical stability than those without drug loading.
Biocompatibility and adhesive properties of PHA microparticles in vitro
In this study mouse fibroblast NIH 3Т3 cells were cultivated on PHA microparticles, and results of using fluorescent DAPI DNA stain, and MTT assay showed that microparticles prepared from PHAs of different chemical compositions did not exhibit cytotoxicity to cells cultured on them and proved to be highly biocompatible. At 3 d after seeding, counts of attached cells showed that the number of cells on microparticles treated with H2O2 plasma was higher. The largest number of cells (up to 28-33 in the field of view) were attached to microparticles prepared from P3HB and P3HB/3HV with 20 mol% 3HV. That number was 1.4 – 1.8 times higher than the number of cells attached to the microparticles sterilized by autoclaving. The number of cells attached to autoclaved microparticles prepared from P3HB/3HV (6.5, 10 and 37 mol% 3HV), P3HB/3HHx, and P3HB/4HB (6.1 and 16 mol% 4HB) was half that recorded on the corresponding microparticles treated with H2O2 plasma. A possible explanation for this might be that treatment of polymer devices by physical methods (laser cutting or plasma) strengthens interphase adhesion joints, increasing surface hydrophilicity and, hence, improving its adhesion properties. MTT assay did not reveal any cytotoxic effect of autoclaved or plasma-treated PHA microparticles. The number of viable cells adhering to the surface of the matrices treated with H2O2 plasma was higher than on the surface of the autoclaved ones in all treatments. On plasma treated microparticles prepared from PHAs with different chemical composition, cells spread well and formed a monolayer. On the corresponding PHA microparticles sterilized by autoclaving, the number of cells was 1.5-2 times lower, and they showed an irregular shape. As differences in the number of cells proliferating on microparticles prepared from PHAs of different types are insignificant, all of the polymers investigated in this study are of good quality, showing high biocompatibility.
Different sized PHBV micro/nanospheres were tested for their effect on cell proliferation using L929 mouse fibroblasts and the MTT test. No significant effect of particle size on proliferation was observed. The effect of particles on cell proliferation was significant when the particle concentration was higher. These cells did not show a change in their shape implying that the decrease is not due to a negative effect like toxicity which could have been observed by light microscopy.
Initial studies of particle penetration into the cells were carried out with the three batches of particles with different sizes. It was show that the cells take up the low and mid nano sized particles. An interesting finding was that the nanospheres were generally located in the cytoplasm near the nuclei. The larger (submicro) particles however, seemed to be unable to penetrate into the cells.
An in vitro study of the inhibiting effect of DOX-loaded microparticles
The cytostatic drug encapsulated in P3HB/3HV microparticles has been proven to be effective against HeLa tumor cells. At implementation of smaller particles (0.2 microns) with the highest load (0.6 µg /ml) the effect of the cytostatic drug depositing is comparable with free form as by the time of beginning of the action, so by the inhibiting effect on the cells. Particles loaded with the medium and lowest concentration (3.2 and 6.0 µg /ml) inhibited the growth of tumor cells only by the 3rd day of the experiment comparable with free DOX, but the beginning of the drug’s action was late in time; the maximum inhibiting effect was observed on the 4th day. This is connected with the kinetics of the drug outflow from the polymer matrix into the culture at which in the first two days the release of the drug in the culture was low (at the level 0,09 µg /ml and 0,07 µg /ml for the highest and the lowest concentration of DOX, correspondingly) and this concentration was insignificant for suppression of HeLa growth.
At implementation of larger polymer particles the effect of DOX depositing was more expressed. Delay in the inhibiting effect was registered only on the first day and only for the lowest and medium concentration of DOX (correspondingly, concentration of DOX in the culture made 0.08 µg /ml and 0.28 µg /ml). Nevertheless, already on the second day the cytostatic effect of the deposited DOX was comparable with the action of the free drug.
These findings demonstrated the efficiency of the cytostatic drug deposited in the microparticles conctrusted from resorbing polymers in relation to the culure of HeLa tumor cells.
Polymeric microcarriers loaded with anti-inflammatory substances in the therapy of experimental skin wounds
Development of effective preparation for the treatment of damaged skin serves as an actual problem of reparative medicine, which is related to a constant increase in the number of skin lesions due to burns, injuries, and surgical treatment.
Here we developed and tested a pharmaceutical form, degradable microparticles with anti-inflammatory substances, for the treatment of experimental skin lesions. We studied the effects of anti-inflammatory substances incorporated in polymeric microparticles made of degradable natural polyhydroxyalkanoate polyesters on experimental skin wounds caused by chemical burns in laboratory animals. Treatment with encapsulated forms of anti-inflammatory substances (applied in gel) accelerated wound healing in comparison with routine therapy (estimated by area of burn wound, wound healing activity, number of acanthotic cells, and number of hair and sebaceous follicles). The results showed the perspectives of usage of developed form of substances (degradable polymeric microparticles) for treatment of skin defects. Microscopy of the damaged skin showed that on days 2-4 of therapy, the histological pattern was similar in all groups. Edema of all dermal layers, inflammatory cell infiltration, disseminated focal necrosis in all dermal layers, and spasm of small vessels were found. Thus, the effects of free and encapsulated agents were similar. However, reparative processes in the skin (area of burn wound, intensity of wound healing, number of acanthosis cords, hair follicles, sebaceous glands, and horny cysts) in animals receiving microparticles were more pronounced. It should be noted that standard ointment compresses with the test agents were put daily for 11 days during the therapy, and applications of microparticles were made once at 3 days. Our results demonstrate positive effects of polymeric PHA-based microparticles with anti-inflammatory agents used as long-acting drugs for restorative treatment of skin lesions.
Study of the Efficiency of Doxorubicin Deposited in PHAs Microparticles on Laboratory Animals with Ehrlich’s Solid Carcinoma
We also have studied the antitumor effects of doxorubicin, encapsulated in PHA matrix, towards Ehrlich’s solid carcinoma (EC). Use of the experimental form of the cytostatic in polymeric microparticles from resorbable PHAs in animals with solid tumor led to inhibition of the cancerous process, comparable to that in response to intravenous free doxorubicin, but without negative effects on the blood system. No negative effect of injection of microparticles charged with doxorubicin was recorded. Blood analysis showed no changes beyond the normal range of values or appreciable differences in comparison with intact animals. Group animals (free doxorubicin) developed a 2-fold reduction of the leukocyte count by the end of experiment. This was presumably caused by the antibiotic toxicity for hemopoietic organs.
Formation of slit-like necroses in tumor tissue was found in all groups after 1 week. In the positive control group, the necroses occupied large fields close to the central part of the tumor, the tumor tissue was retained at the periphery, in zones of infiltrative growth. In experimental groups, the necroses were grouped in large fields in the central compartments, while smaller ones were chaotically scattered at the periphery. The area of necrotic zones in experimental and positive control groups was about 30% of tumor tissue area. Two weeks after the beginning of therapy the antitumor effect was significantly higher in the positive control group than in experimental groups. However, after 3 weeks the maximum tumor growth inhibition (area of necrotic zones reaching 78% of tumor tissue area) was recorded in experimental group 5 (in response to double cycle dose of the drug). In group 3 animals (intravenous drug weekly) and in group 4 (a single cycle dose of the drug encapsulated in microparticles) the necrotic zones occupied 63-64% of tumor tissue area during this period; the values in these groups virtually did not differ. It is noteworthy that the necrotic zone area in the negative control group varied from 18 to 30% throughout the entire experiment.
Our experiments on the solid EC model demonstrated that PHAs was fit to serve as a doxorubicin carrier for local treatment and showed the antitumor efficiency of the drug encapsulated PHAs microparticles.