Characteristics of Microparticles Based on Resorbable Polyhydroxyalkanoates Loaded with Antibacterial and Cytostatic Drugs

Abstract

The development of controlled drug delivery systems, in the form of microparticles, is an important area of experimental pharmacology. The success of the design and the quality of the obtained microparticles are determined by the method of manufacture and the properties of the material used as a carrier. The goal is to obtain and characterize microparticles depending on their method of preparation, the chemical composition of the polymer and the load of the drugs. To obtain microparticles, four types of degradable PHAs, differing in their chemical compositions, degrees of crystallinity, molecular weights and temperature characteristics, were used (poly-3-hydroxybutyrate and copolymers 3-hydroxybutyric-co-3-hydroxyvaleric acid, 3-hydroxybutyric-co-4-hydroxybutyric acid, and 3-hydroxybutyric-co-3-hydroxyhexanoic acid). The characteristics of microparticles from PHAs were studied. Good-quality particles with an average particle diameter from 0.8 to 65.0 μm, having satisfactory ζ potential values (from -18 to -50 mV), were obtained. The drug loading content, encapsulation efficiency and in vitro release were characterized. Composite microparticles based on PHAs with additives of polyethylene glycol and polylactide-co-glycolide, and loaded with ceftriaxone and 5-fluorouracil, showed antibacterial and antitumor effects in E. coli and HeLa cultures. The results indicate the high potential of PHAs for the design of modern and efficient drug delivery systems.

Keywords: E. coli and HeLa cultures; biodegradable polyhydroxyalkanoates; drug delivery systems; drug efficiency; microparticles; properties.

Figure 1

Characteristics of microparticles from P(3HB) obtained using various methods: (A)—SEM image of microparticles; (B) size distribution, mean diameter (μm) and ζ potential (mV) of microparticles. Emulsion was mixed at speeds of 500 rpm (a), 1000 rpm (b), 15,000 rpm (c), and 24,000 rpm (d); spray-drying process parameters were as follows: feed pump speed and temperature 75 °C, 1.5 mL/min (e), 95 °C, 3.2 mL/min (f); the red arrow indicates the burst particle. The scale bar is 40 μm (a,b), 5 μm (c,d), and 30 μm (e,f).

Figure 2

Characteristics of PHA microparticles of various chemical compositions and in compositions with PEG and PLGA: (A) SEM image of microparticles; (B) size distribution, mean diameter (μm) and ζ potential of microparticles (mV). The chemical compositions of microparticles as follows: P(3HB/3HV) 93.2/6.8 (a), P(3HB/4HHx) 86.4/13.6 (b), P(3HB/4HB) 84.0/16.0 (c), P3HB/PEG 75/25 (d), and P3HB/PLGA 75/25 (e). The scale bar is 5 μm.

Figure 3

Characteristics of drug-loaded microparticles from PHAs and in composition with PEG and PLGA obtained using the emulsion method and spray-drying method: (A) SEM image of microparticles; (B) drug release from PHAs microparticles, and average diameter (μm) and ζ potential (mV) of microparticles. SEM image, ζ potential and average diameter are given for P3HB microparticles containing 10% ceftriaxone (a), P(3HB/3HV) 67.2/32.8 containing 5% doxorubicin (b), P(3HB/4HHx) 86.4/13.6 containing 5% doxorubicin (c), P(3HB/4HB) 84.0/16.0 containing 5% doxorubicin (d), P3HB microparticles containing 5% 5-fluorourocil (e); the red arrow indicates the drug, P3HB/PEG 75/25 containing 5% 5-fluorourocil (f), and P3HB/PLGA 75/25 containing 5% rifampicin (g). The scale bar is 5 μm (a–d) and 2 μm (e–g).

Figure 4

The antibacterial efficacy of drug-loaded microparticles in E. coli culture: rifampicin-loaded microparticles ((a) commercial disc, (b) P(3HB), (c) P(3HB)/PEG, (d) P(3HB)/PLGA) and ceftriaxone-loaded microparticles. (A) Microparticles prepared using solvent evaporation method. (B) Microparticles obtained using mini spray dryer ((e,h) commercial disc; (f,i) P(3HB)/PEG; (g,j) P(3HB)).

Figure 5

(A) Fluorescent staining of HeLa cells treated with 5-FLU ((a) “−” Control (intact cells); (b) “+” Control (free drug); (c)—P(3HB)/5-Fluorouracil; (d)—P(3HB)/PEG /5-fluorouracil; (B) MTT assay. Morphology of viable cells and nonviable cells when stained by acridine orange/ethidium bromide: viable cells stain uniformly green (a); early-apoptotic cells with intact plasma membranes appear light green, with condensed chromatin (e); apoptotic cells are stained bright green-orange because membrane deformation starts to occur, and EB can enter the cell (b–d); and late-apoptotic nonviable cells are stained bright orange or red because of the entry of ethidium bromide into these cells (f–h). The scale bar 10 μm (24 h) and 100 μm (72 h).

Figure 6

Schematic representation of methods for obtaining PHA microparticles: emulsion method (A), and spray drying (B).