PLGA-based microparticles loaded with bacterial-synthesized prodigiosin for anticancer drug release: Effects of particle size on drug release kinetics and cell viability.

This paper presents the synthesis and physicochemical characterization of biodegradable poly (d,l-lactide-co-glycolide) (PLGA)-based microparticles that are loaded with bacterial-synthesized prodigiosin drug obtained from Serratia marcescens subsp. Marcescens bacteria for controlled anticancer drug delivery. The micron-sized particles were loaded with anticancer drugs [prodigiosin (PG) and paclitaxel (PTX) control] using a single-emulsion solvent evaporation technique. The encapsulation was done in the presence of PLGA (as a polymer matrix) and poly-(vinyl alcohol) (PVA) (as an emulsifier). The effects of processing conditions (on the particle size and morphology) are investigated along with the drug release kinetics and drug-loaded microparticle degradation kinetics. The localization and apoptosis induction by prodigiosin in breast cancer cells is also elucidated along with the reduction in cell viability due to prodigiosin release. The implication of this study is for the potential application of prodigiosin PLGA-loaded microparticles for controlled delivery of cancer drug and treatment to prevent the regrowth or locoregional recurrence, following surgical resection of triple negative breast tumor.

Polyactives: controlled and sustained bioactive release via hydrolytic degradation.

Significant and promising advances have been made in the polymer field for controlled and sustained bioactive delivery. Traditionally, small molecule bioactives have been physically incorporated into biodegradable polymers; however, chemical incorporation allows for higher drug loading, more controlled release, and enhanced processability. Moreover, the advent of bioactive-containing monomer polymerization and hydrolytic biodegradability allows for tunable bioactive loading without yielding a polymer residue. In this review, we highlight the chemical incorporation of different bioactive classes into novel biodegradable and biocompatible polymers. The polymer design, synthesis, and formulation are summarized in addition to the evaluation of bioactivity retention upon release via in vitro and in vivo studies.

Salicylic acid-based poly(anhydride esters) for control of biofilm formation in Salmonella enterica serovar Typhimurium.

AIMS: Bacterial biofilms generally are more resistant to stresses as compared with free planktonic cells. Therefore, the discovery of antimicrobial stress factors that have strong inhibitory effects on bacterial biofilm formation would have great impact on the food, personal care, and medical industries. METHODS AND RESULTS: Salicylate-based poly(anhydride esters) (PAE) have previously been shown to inhibit biofilm formation, possibly by affecting surface attachment. Our research evaluated the effect of salicylate-based PAE on biofilm-forming Salmonella enterica serovar Typhimurium. To remove factors associated with surface physical and chemical parameters, we utilized a strain that forms biofilms at the air-liquid interface. Surface properties can influence biofilm characteristics, so the lack of attachment to a solid surface eliminates those constraints. The results indicate that the salicylic acid-based polymers do interfere with biofilm formation, as a clear difference was seen between bacterial strains that form biofilms at the air-liquid interface (top-forming) and those that form at the surface-liquid interface (bottom-forming). CONCLUSION: These results lead to the conclusion that the polymers may not interfere with attachment; rather, the polymers likely affect another mechanism essential for biofilm formation in Salmonella. SIGNIFICANCE AND IMPACT OF THE STUDY: Biofilm formation can be prevented through controlled release of nature-derived antimicrobials formulated into polymer systems.

Microcontact printing of proteins on oxygen plasma-activated poly(methyl methacrylate).

This paper describes a method for microcontact printing protein solutions onto polymer substrates temporarily activated by oxygen plasma. Following plasma treatment, poly(dimethyl siloxane) (PDMS) stamps were coated with an aqueous laminin solution then placed in direct contact with plasma-treated poly(methyl methacrylate) (PMMA) substrates. This process resulted in well defined laminin stripes on the PMMA surface when printing was performed within 45min of the plasma treatment. Axonal outgrowth from embryonic chick dorsal root ganglia (DRG) was largely confined to the stamped pattern, while over 90% of primary rat Schwann cells adhered to the protein stamped areas on the PMMA substrates. Oxygen-plasma treatment of the PMMA surface was necessary to deposit proteins that direct axonal outgrowth from chick DRG and Schwann cell adherence.

Cytotoxicity of a unimolecular polymeric micelle and its degradation products.

The cytotoxicity of a unimolecular polymeric micelle (1) and its degradation products was assessed by cell proliferation and viability with L929 mouse areolar/adipose fibroblasts. Polymer 1 and poly(ethylene glycol) (PEG) were diluted to concentrations from 10(-4) to 10(-6) M in culture media, whereas the degradation products were diluted to concentrations (10(-4) to 10(-6) M) that would theoretically occur upon degradation of polymer 1. The polymer degradation products that were evaluated included mucic acid, hexanoic acid, 1,1,1-tris(4-hydroxyphenyl)ethane (THPE),2,3,4,5-tetrakis-hexanoyloxy-hexanedioic acid or MA(hex), Core(hex), and PEG5, which is PEG of molecular weight 5000. Cells exposed to polymer 1 proliferated at the same rate as cells grown in polymer-free or PEG5-containing solutions up to 36 h. In both the polymer 1 and PEG5 solutions, cytotoxicity was not observed at any concentration (up to 10(-4) M) as indicated by cell attachment, growth, and morphology. Fibroblasts exposed to the degradation products fared as well as fibroblasts in contact with polymer 1 and PEG5, except for cells exposed to the highest concentration (10(-4) M) of THPE.

Regional drug delivery with radiation for the treatment of Ewing's sarcoma. In vitro development of a taxol release system.

Recently, several studies have suggested the radiosensitizing effect of taxol, a microtubular inhibitor. Our overall hypothesis is that a combination of radiation and taxol may demonstrate therapeutic efficacy over doses of either individually. Studies examining taxol use have mostly focused on systemic administration, which can lead to undesired effects. To circumvent these side effects, we propose a locally administered polymeric microsphere delivery system combined with radiation therapy for the treatment of Ewing's sarcoma. The present study focuses on the in vitro ability of taxol when present as a microencapsulated drug delivery system, and delivered locally at the site of the sarcoma/tumor, to block cells in the G2/M phase of the cell cycle and potentially enhance the radiation sensitivity of cells. Using the bioresorbable poly(anhydride-co-imide), poly[pyromellityl-imidoalanine-1,6-bis(carboxy-phenoxy)hexane] (PMA-CPH), and the radiosensitizing agent taxol, a microsphere based delivery system was fabricated. A solvent evaporation technique was used to encapsulate taxol at doses of 1%, 5%, and 10% in PMA-CPH microspheres. Release kinetics studies demonstrated that the total amount of taxol released and the release rate were directly dependent on loading percentage. Taxol's bioactivity and radiosensitizing ability were measured using flow cytometry. Co-culture of Ewing's sarcoma cells with and without taxol-loaded microspheres demonstrated that released taxol retained its bioactivity and effectively blocked cells in the radiosensitive G2/M phase of mitosis. The taxol-radiation delivery system studied achieved an 83% decrease in tumor cell count compared to control. Taxol effectively sensitized Ewing's sarcoma cells to radiation with radiosensitivity shown to be independent of radiation dose at levels of dosages studied. This work has demonstrated that taxol can be effectively released from a biodegradable PMA-CPH microsphere delivery system while maintaining potent combined cytotoxic and radiosensitizing abilities.

Degradable poly(anhydride ester) implants: effects of localized salicylic acid release on bone.

Degradable poly(anhydride ester) implants in which the polymer backbone breaks down into salicylic acid (SA) were investigated. In this preliminary work, local release of SA from the poly(anhydride esters), thus classified as 'active polymers', on healthy bone and tissue was evaluated in vivo using a mouse model. Degradable polyanhydrides that break down into inactive by-products were used as control membranes because of their chemical similarity to the active polymers. Small polymer squares were inserted over the exposed palatal bone adjacent to the maxillary first molars. Active polymer membranes were placed on one side of the mouth, control polymers placed on the contra lateral side. Intraoral clinical examination showed that active polymer sites were less swollen and inflamed than control polymer sites. Histopathological examination at day 1 showed essentially no difference between control and active polymers. After 4 days, active polymer sites showed epithelial proliferation to a greater extent than the polyanhydride controls. After 20 days, active polymer sites showed greater thickness of new palatal bone and no resorptive areas, while control polymer sites showed less bone thickness as well as resorption including lacunae involving cementum and dentine. From these preliminary studies, we conclude that active polymers, namely poly(anhydride esters), stimulated new bone formation.

Synthesis and degradation characteristics of salicylic acid-derived poly(anhydride-esters).

A biodegradable poly(anhydride-ester) was synthesized by melt condensation polymerization of the acetylated monomer to yield a novel polymeric prodrug. The polymer we have synthesized is composed of alkyl chains linked by ester bonds to aromatic moieties, specifically salicylic acid--the active component of aspirin. With the medicinal properties attributed to salicylic acid and the ease of metabolism, the incorporation of this compound into a polymer backbone yields a polymeric prodrug that may have potential in a variety of applications (i.e., inflammatory bowel disease). For these reasons, we have designed a synthetic scheme that yields the desired poly(anhydride-ester). The in vitro hydrolytic degradation of these polymers has been performed and results indicate that the polymer degradation rate is pH-dependent.

Proliferation, morphology, and protein expression by osteoblasts cultured on poly(anhydride-co-imides).

In vitro cell biocompatibility models are crucial in the study of any newly synthesized material. Our focus has been on the development of a new class of biocompatible, degradable, high-strength polymeric materials, the poly(anhydride-co-imides), for use in bone regeneration. This study examined osteoblast cell adherence, proliferation, viability, and phenotypic preservation on the surface of the poly(anhydride-co-imide) poly[pyromellitylimidoalanine (PMA-ala):1,6-bis(carboxyphenoxy) hexane (CPH)] over a period of time. Cell proliferation on PMA-ala:CPH degradable matrices over 21 days was examined. Throughout the 21-day period of study, osteoblast proliferation was similar on PMA-ala:CPH and on tissue culture polystyrene controls. Osteoblasts maintained their characteristic morphology as demonstrated by both scanning electron microscopy and immunofluorescence studies. Alkaline phosphatase activity for cells grown on PMA-ala:CPH was confirmed. Retention of the osteoblastic phenotype was demonstrated using immunofluorescence techniques and staining with antibodies against osteocalcin (an extracellular matrix protein of bone) and osteopontin (a marker of cell adhesion). Radioimmunoassay results provided evidence that levels of osteocalcin production by osteoblasts were similar when cells were cultured on PMA-ala:CPH and on tissue culture polystyrene controls. The present study provided evidence of normal osteoblast function on PMA-ala:CPH surfaces. PMA-ala:CPH may therefore be useful as a synthetic material for orthopedic applications.

Preliminary in vivo report on the osteocompatibility of poly(anhydride-co-imides) evaluated in a tibial model.

A novel class of polymers with mechanical properties similar to cancellous bone are being investigated for their ability to be used in weight-bearing areas for orthopedic applications. The poly(anhydride-co-imide) polymers based on poly[trimellitylimidoglycine-co-1,6-bis(carboxyphenoxy)hexan e] (TMA-Gly:CPH) and poly[pyromellitylimidoalanine-co-1,6-bis(carboxyphenoxy)hexa ne] (PMA-Ala:CPH) in molar ratios of 30:70 were investigated for osteocompatibility, with effects on the healing of unicortical 3-mm defects in rat tibias examined over a 30-day period. Defects were made with surgical drill bits (3-mm diameter) and sites were filled with poly(anhydride-co-imide) matrices and compared to the control poly(lactic acid-glycolic acid) (PLAGA) (50:50), a well-characterized matrix frequently used in bone regeneration studies, and defects without polymeric implants. At predetermined time intervals (3, 6, 9, 12, 20, and 30 days), animals were sacrificed and tissue histology was examined for bone formation, polymer-tissue interaction, and local tissue response by light microscopy. The studies revealed that matrices of TMA-Gly:CPH and PMA-Ala:CPH produced responses similar to the control PLAGA with tissue compatibility characterized by a mild response involving neutrophils, macrophages, and giant cells throughout the experiment for all matrices studied. Matrices of PLAGA were nearly completely degraded by 21 days in contrast to matrices of TMA-Gly:CPH and PMA-Ala:CPH that displayed slow erosion characteristics and maintenance of shape. Defects in control rats without polymer healed by day 12, defects containing PLAGA healed after 20 days, and defects containing poly(anhydride-co-imide) matrices produced endosteal bone growth as early as day 3 and formed bridges of cortical bone around matrices by 30 days. In addition, there was marrow reconstitution at the defect site for all matrices studied along with matured bone-forming cells. This study suggests that novel poly(anhydride-co-imides) are promising polymers that may be suitable for use as implants in bone surgery, especially in weight-bearing areas.

Chemical changes during in vivo degradation of poly(anhydride-imide) matrices.

The in vivo degradation characteristics of a novel class of biodegradable polymers, poly(anhydride-imides), were investigated. The poly(anhydride-imides) examined were poly[trimellitylimidoglycine-co-1,6-bis(p-carboxyphenoxy)hex ane] (TMA-gly:CPH) in 10:90, 30:70 and 50:50 molar ratios and poly[pyromellitylimidoalanine-co-1,6-bis(p-carboxyphenoxy)he xane] (PMA-ala:CPH) in 10:90 and 30:70 molar ratios. The polymer matrices were compression-molded into circular discs, then implanted in rat subcutaneous tissues for nearly two months. At defined time intervals, the animals were sacrificed and explants analyzed. Proton NMR spectroscopic analysis revealed a complete absence of imide monomer units in PMA-ala: CPH compositions after 28 d and complete removal of imide units at 56 d from TMA-gly matrices. Gross observation of the implants closely correlated to the imide content: with decreasing imide content, the explants darkened and fragmented at a faster rate. The chemical compositions of the poly(anhydride imide) explants were also monitored using IR spectroscopy. The residual amount of anhydride bonds in the polymer backbone following implantation were calculated from peaks specific to the anhydride bonds relative to the total amount of carbonyl bonds present. Initially, the imide (TMA-gly or PMA-ala) anhydride bonds were rapidly hydrolyzed then solubilized, followed by the slower hydrolysis of the CPH monomer anhydride bonds.

Poly(anhydride-co-imides): in vivo biocompatibility in a rat model.

The degradation and tissue compatibility characteristics of a novel class of biodegradable poly(anhydride-co-imide) polymers: poly[trimellitylimidoglycine-co-1,6-bis(carboxyphenoxy)hexan e] (TMA-gly: CPH) (in 10:90; 30:70 and 50: 50 molar ratios) and poly[pyromellitylimidoalanine-co-1,6-bis(carboxyphenoxy)hexa ne] (PMA-ala:CPH) (in 10:90 and 30:70 molar ratios) were investigated and compared with control poly(lactic acid/glycolic acid) (PLAGA in 50:50 molar ratio) matrices, a well-characterized biocompatible polymer, in rat subcutaneous tissues for 60 days. Polymers were compression-molded into circular discs of 14 mm x 1 mm in diameter. On post-operative days 7, 14, 28 and 60, histological tissue samples were removed, prepared by fixation and staining, and analyzed by light microscopy. PLAGA matrices produced mild inflammatory reactions and were completely degraded at the end of 60 days, leaving implant tissues that were similar to surgical wounds without implants. TMA-gly:CPH (10:90 and 30:70) matrices produced mild inflammatory reactions by the end of 60 days, similar to those seen with PLAGA. TMA-gly: CPH (50: 50) produced moderate inflammatory reactions characterized by macrophages and edema. PMA-ala:CPH matrices elicited minimal inflammatory reactions that were characterized by fibrous encapsulation by the end of 60 days. In vivo degradation rates of poly(anhydride-co-imides) were similar to PLAGA. Both PMA-ala:CPH and TMA-gly: CPH matrices maintained their shapes and degraded at a constant rate over the period of two months. These polymers, possessing good mechanical properties and tissue compatibility, may be useful in weight-bearing applications in bone.

In vitro bone biocompatibility of poly (anhydride-co-imides) containing pyromellitylimidoalanine.

Poly(anhydride-co-imides) are currently under study for applications involving bone. The cytotoxicity of a series of poly(anhydride-co-imides) with osteoblast-like cells (MC3T3-E1) was evaluated. The imide component of the copolymers was based on pyromellitylimidoalanine and the anhydride component was based on either sebacic acid or 1,6-bis(carboxyphenoxy)hexane. Cell adhesion and proliferation on the surfaces of the polymer discs were observed by environmental scanning electron microscopy. During the first 24 hours of attachment, the cells showed normal morphology when cultured on copolymers containing 1,6-bis(carboxyphenoxy)hexane. The cells did not adhere to the polymers containing sebacic acid, probably due to the rapid degradation of the polymer surfaces. Concurrently, the effects of polymer breakdown products on osteoblast-like cells were evaluated by studying their proliferation (cell numbers), viability (dye exclusion), morphology (light microscopy), and phenotypic expression. The morphology of osteoblast-like cells cultured in the presence of the polymer breakdown products pyromellitylimidoalanine and pyromellitic acid was found to be similar to that of the same cells grown on tissue culture polystyrene and consisted of a characteristic polygonal shape. With use of a monoclonal antibody to osteocalcin, these cells were shown to demonstrate preserved osteoblast phenotype with growth over a 21-day period. In addition, the cells reached confluency after 3-4 days, similar to cells grown on tissue culture polystyrene. This in vitro evaluation showed that the poly(anhydride-co-imides) evaluated are non-cytotoxic and may be viable biomaterials for orthopaedic applications.

Cytotoxicity testing of poly(anhydride-co-imides) for orthopedic applications.

The cytotoxicity of a series of poly(anhydride-co-imides) with osteoblast-like cells (MC3T3-E1) was evaluated. The imide component of the copolymers was based on trimellitylimidoglycine (TMA-gly), and the anhydride component was based on either sebacic acid (SA) or 1,6-bis(carboxyphenoxy)hexane (CPH). Cell adhesion and proliferation on surfaces of the polymer discs were observed by environmental scanning electron microscopy (ESEM). During the first 24 h of attachment, cells showed normal morphology when cultured on the various copolymers of CPH. Concurrently, the effects of polymer breakdown products on osteoblast-like cells were evaluated by studying their proliferation (cell numbers), viability (dye exclusion), and morphology (light microscopy). Cell cultures in the presence of these breakdown products resulted in normal morphologies and reached confluency after 7 days. This initial in vitro evaluation with osteoblast-like cells suggests that the poly(anhydride-co-imides) may be viable carriers for osteoblasts.