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Poly(lactic-co-glycolic acid) (PLGA) is widely used as a vehicle for delivery of pharmaceutically relevant payloads. PLGA is readily fabricated as a nano- or microparticle (MP) matrix to load both hydrophobic and hydrophilic small molecular drugs as well as biomacromolecules such as nucleic acids and proteins. However, targeting such payloads to the cell cytosol is often limited by MP entrapment and degradation within acidic endolysosomes. Poly(propylacrylic acid) (PPAA) is a polyelectrolyte polymer with the membrane disruptive capability triggered at low pH. PPAA has been previously formulated in various carrier configurations to enable cytosolic payload delivery, but requires sophisticated carrier design. Taking advantage of PPAA functionality, we have incorporated PPAA into PLGA MPs as a simple polymer mixture to enhance cytosolic delivery of PLGA-encapsulated payloads. Rhodamine loaded PLGA and PPAA/PLGA blend MPs were prepared by a modified nanoprecipitation method. Incorporation of PPAA into PLGA MPs had little to no effect on the size, shape, or loading efficiency, and evidenced no toxicity in Chinese hamster ovary epithelial cells. Notably, incorporation of PPAA into PLGA MPs enabled pH-dependent membrane disruption in a hemolysis assay, and a three-fold increased endosomal escape and cytosolic delivery in dendritic cells after 2 h of MP uptake. These results demonstrate that a simple PLGA/PPAA polymer blend is readily fabricated into composite MPs, enabling cytosolic delivery of an encapsulated payload. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1022-1033, 2018.
© 2017 Wiley Periodicals, Inc.
The present study investigated the immunoenhancing property of our newly designed nanovaccine, that is, its ability to induce antigen-specific immunity. This study also evaluated the synergistic effect of a novel compound PBS-44, an α-galactosylceramide analog, in boosting the immune response induced by our nanovaccine. The nanovaccine was prepared by encapsulating ovalbumin (ova) and an adjuvant within the poly(lactic-co-glycolic acid) nanoparticles. Quantitative analysis of our study data showed that the encapsulated vaccine was physically and biologically stable; the core content of our nanovaccine was found to be released steadily and slowly, and nearly 90% of the core content was slowly released over the course of 25 days. The in vivo immunization studies exhibited that the nanovaccine induced stronger and longer immune responses compared to its soluble counterpart. Similarly, intranasal inhalation of the nanovaccine induced more robust antigen-specific CD8 T cell response than intraperitoneal injection of nanovaccine.
Biodegradable tissue engineering scaffolds are commonly fabricated from poly(lactide-co-glycolide) (PLGA) or similar polyesters that degrade by hydrolysis. PLGA hydrolysis generates acidic breakdown products that trigger an accelerated, autocatalytic degradation mechanism that can create mismatched rates of biomaterial breakdown and tissue formation. Reactive oxygen species (ROS) are key mediators of cell function in both health and disease, especially at sites of inflammation and tissue healing, and induction of inflammation and ROS are natural components of the in vivo response to biomaterial implantation. Thus, polymeric biomaterials that are selectively degraded by cell-generated ROS may have potential for creating tissue engineering scaffolds with better matched rates of tissue in-growth and cell-mediated scaffold biodegradation. To explore this approach, a series of poly(thioketal) (PTK) urethane (PTK-UR) biomaterial scaffolds were synthesized that degrade specifically by an ROS-dependent mechanism. PTK-UR scaffolds had significantly higher compressive moduli than analogous poly(ester urethane) (PEUR) scaffolds formed from hydrolytically-degradable ester-based diols (p < 0.05). Unlike PEUR scaffolds, the PTK-UR scaffolds were stable under aqueous conditions out to 25 weeks but were selectively degraded by ROS, indicating that their biodegradation would be exclusively cell-mediated. The in vitro oxidative degradation rates of the PTK-URs followed first-order degradation kinetics, were significantly dependent on PTK composition (p < 0.05), and correlated to ROS concentration. In subcutaneous rat wounds, PTK-UR scaffolds supported cellular infiltration and granulation tissue formation, followed first-order degradation kinetics over 7 weeks, and produced significantly greater stenting of subcutaneous wounds compared to PEUR scaffolds. These combined results indicate that ROS-degradable PTK-UR tissue engineering scaffolds have significant advantages over analogous polyester-based biomaterials and provide a robust, cell-degradable substrate for guiding new tissue formation.
Copyright © 2014 Elsevier Ltd. All rights reserved.
Electrospun meshes suffer from poor cell infiltration and limited thickness, which restrict their use to thin tissue applications. Herein, we demonstrate two complementary processes to overcome these limitations and achieve elastomeric composites that may be suitable for ligament repair. First, C3H10T1/2 mesenchymal stem cells were incorporated into electrospun meshes using a hybrid electrospinning/electrospraying process. Second, electrospun meshes were rolled and formed into composites with an interpenetrating polyethylene glycol (PEG) hydrogel network. Stiffer composites were formed from poly(lactic-co-glycolic acid) (PLGA) meshes, while softer and more elastic composites were formed from poly(ester-urethane urea) (PEUUR) meshes. As-spun PLGA and PEUUR rolled meshes had tensile moduli of 19.2 ± 1.9 and 0.86 ± 0.34 MPa, respectively, which changed to 11.6 ± 4.8 and 1.05 ± 0.39 MPa with the incorporation of a PEG hydrogel phase. In addition, cyclic tensile testing indicated that PEUUR-based composites deformed elastically to at least 10%. Finally, C3H10T1/2 cells incorporated into electrospun meshes survived the addition of the PEG phase and remained viable for up to 5 days. These results indicate that the fabricated cellularized composites are support cyclic mechanical conditioning, and have potential application in ligament repair.
Nonunion is a common complication in open fractures and other severe bone injuries. Recombinant human bone morphogenetic protein-2 (rhBMP-2) delivered on a collagen sponge enhances healing of fractures. However, the burst release of rhBMP-2 necessitates supra-physiological doses of rhBMP-2 to achieve a robust osteogenic effect, which introduces risk of ectopic bone formation and severe inflammation and increases the cost. Although the concept that the ideal pharmacokinetics for rhBMP-2 includes both a burst and sustained release is generally accepted, investigations into the effects of the release kinetics on new bone formation are limited. In the present study, biodegradable polyurethane (PUR) and PUR/microsphere [PUR/poly(lactic-co-glycolic acid)] composite scaffolds with varying rhBMP-2 release kinetics were compared to the collagen sponge delivery system in a critical-sized rat segmental defect model. Microcomputed tomography analysis indicated that a burst followed by a sustained release of rhBMP-2 from the PUR scaffolds regenerated 50% more new bone than the collagen sponge loaded with rhBMP-2, whereas a sustained release without the burst did not form significantly more bone than the scaffold without rhBMP-2. This study demonstrated that the putative optimal release profile (i.e., burst followed by sustained release) for rhBMP-2 can be achieved using PUR scaffolds, and that this enhanced pharmacokinetics regenerated more bone than the clinically available standard of care in a critical-sized defect in rat femora.
Technologies to increase tissue vascularity are critically important to the fields of tissue engineering and cardiovascular medicine. Currently, limited technologies exist to encourage angiogenesis and arteriogenesis in a controlled manner. In the present study, we describe an injectable controlled release system consisting of VEGF encapsulated in poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs). The majority of VEGF was released gradually over 2-4 days from the NPs as determined by an ELISA release kinetics experiment. An in vitro aortic ring bioassay was used to verify the bioactivity of VEGF-NPs compared with empty NPs and no treatment. A mouse femoral artery ischemia model was then used to measure revascularization in VEGF-NP-treated limbs compared with limbs treated with naked VEGF and saline. 129/Sv mice were anesthetized with isoflurane, and a region of the common femoral artery and vein was ligated and excised. Mice were then injected with VEGF-NPs, naked VEGF, or saline. After 4 days, three-dimensional microcomputed tomography angiography was used to quantify vessel growth and morphology. Mice that received VEGF-NP treatment showed a significant increase in total vessel volume and vessel connectivity compared with 5 microg VEGF, 2.5 microg VEGF, and saline treatment (all P < 0.001). When the yield of the fabrication process was taken into account, VEGF-NPs were over an order of magnitude more potent than naked VEGF in increasing blood vessel volume. Differences between the VEGF-NP group and all other groups were even greater when only small-sized vessels under 300 mum diameter were analyzed. In conclusion, sustained VEGF delivery via PLGA NPs shows promise for encouraging blood vessel growth in tissue engineering and cardiovascular medicine applications.
A key tenet of tissue engineering is the principle that the scaffold can perform the dual roles of biomechanical and biochemical support through presentation of the appropriate mediators to surrounding tissue. While growth factors have been incorporated into scaffolds to achieve sustained release, there are a limited number of studies investigating release of biologically active molecules from reactive two-component polymers, which have potential application as injectable delivery systems. In this study, we report the sustained release of platelet-derived growth factor (PDGF) from a reactive two-component polyurethane. The release of PDGF was bi-phasic, characterized by an initial burst followed by a period of sustained release for up to 21 days. Despite the potential for amine and hydroxyl groups in the protein to react with the isocyanate groups in the reactive polyurethane, the in vitro bioactivity of the released PDGF was largely preserved when added as a lyophilized powder. PUR/PDGF scaffolds implanted in rat skin excisional wounds accelerated wound healing relative to the blank PUR control, resulting in almost complete healing with reepithelization at day 14. The presence of PDGF attracted both fibroblasts and mononuclear cells, significantly accelerating degradation of the polymer and enhancing formation of new granulation tissue as early as day 3. The ability of reactive two-component PUR scaffolds to promote new tissue formation in vivo through local delivery of PDGF may present compelling opportunities for the development of novel injectable therapeutics.
This work evaluates various techniques for the incorporation of poly(ethylene glycol) (PEG) onto biodegradable nanoparticles (NPs) of poly(lactic-co-glycolic acid) (PLGA) or poly(lactic acid) (PLA) with the purpose of providing a functional site for surface conjugation of targeting agents and for improving surface properties. The techniques compared were based on NP preparation with blends of PLGA and poloxamer or with block copolymers of PLGA/PLA with PEG. Blending of PLGA with poloxamer 407 resulted in the incorporation of the latter to up to a 43 wt % content. Direct conjugation of heterofunctional NH2-PEG-COOH to the surface of premade NPs was not highly effective. Preparation of copolymers of PLGA with PEG was determined to be more effective and versatile by polymerization of lactide and glycolide dimers onto the hydroxyl group of heterofunctional OH-PEG-COOH than by conjugation of the premade polymers with carbodiimide chemistry. NPs prepared with these copolymers confirmed the surface localization of PEG and proved to be useful for conjugation of mouse immumoglobulin as a model targeting agent.
Even though degradation products of biodegradable polymers are known to be largely non-cytotoxic, little detailed information is available regarding the degradation rate-dependent acidic byproduct effect of the scaffold. In vitro and in vivo scaffold degradation rate could be differentiated using a fast degrading polymer (e.g., poly D, L-lactic-glycolic acid co-polymer, PLGA, 50:50) and a slow degrading polymer (e.g., poly epsilon-caprolactone, PCL). We applied a new method to develop uniform 10 microm thickness of high porous scaffolds using a computer-controlled knife coater with a motion stage and exploiting phase transition properties of a combination of salts and water in salt-leaching method. We then verified in vitro the effect of fast degradation by assessing the viability of primary mouse aortic smooth muscle cell cultured in the three-dimensional scaffolds. We found that cell viability was inversely related to degradation rate and was dependent on the depth from the seeding (upper) surface toward the lower surface. The pH measurement of culture medium using fluorescence probes showed time-dependent decrease in pH in the PLGA scaffolds, corresponding to PLGA degradation, and closely related to cell viability. In vivo analysis of scaffolds implanted subcutaneously into the back of mice, showed significant differences in inflammation and cell invasion into PLGA vs. PCL. Importantly, these were correlated with the degree of the functional angiogenesis within the scaffolds. Again, PLGA scaffolds demonstrated less cell mobilization and less angiogenesis, further supporting the negative effect of the acidic environment created by the degradation of biocompatible polymers.