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1.
Mol Ther Methods Clin Dev ; 25: 491-507, 2022 Jun 09.
Article in English | MEDLINE | ID: mdl-35615709

ABSTRACT

Duchenne muscular dystrophy (DMD) is a muscle wasting disorder caused by mutations in the DMD gene. Restoration of full-length dystrophin protein in skeletal muscle would have therapeutic benefit, but lentivirally mediated delivery of such a large gene in vivo has been hindered by lack of tissue specificity, limited transduction, and insufficient transgene expression. To address these problems, we developed a lentiviral vector, which contains a muscle-specific promoter and sequence-optimized full-length dystrophin, to constrain dystrophin expression to differentiated myotubes/myofibers and enhance the transgene expression. We further explored the efficiency of restoration of full-length dystrophin in vivo, by grafting DMD myoblasts that had been corrected by this optimized lentiviral vector intramuscularly into an immunodeficient DMD mouse model. We show that these lentivirally corrected DMD myoblasts effectively reconstituted full-length dystrophin expression in 93.58% ± 2.17% of the myotubes in vitro. Moreover, dystrophin was restored in 64.4% ± 2.87% of the donor-derived regenerated muscle fibers in vivo, which were able to recruit members of the dystrophin-glycoprotein complex at the sarcolemma. This study represents a significant advance over existing cell-mediated gene therapy strategies for DMD that aim to restore full-length dystrophin expression in skeletal muscle.

2.
Mol Ther Nucleic Acids ; 28: 261-278, 2022 Jun 14.
Article in English | MEDLINE | ID: mdl-35433111

ABSTRACT

We investigated the feasibility of utilizing an exon-skipping approach as a genotype-dependent therapeutic for neurofibromatosis type 1 (NF1) by determining which NF1 exons might be skipped while maintaining neurofibromin protein expression and GTPase activating protein (GAP)-related domain (GRD) function. Initial in silico analysis predicted exons that can be skipped with minimal loss of neurofibromin function, which was confirmed by in vitro assessments utilizing an Nf1 cDNA-based functional screening system. Skipping of exons 17 or 52 fit our criteria, as minimal effects on protein expression and GRD activity were noted. Antisense phosphorodiamidate morpholino oligomers (PMOs) were utilized to skip exon 17 in human cell lines with patient-specific pathogenic variants in exon 17, c.1885G>A, and c.1929delG. PMOs restored functional neurofibromin expression. To determine the in vivo significance of exon 17 skipping, we generated a homozygous deletion of exon 17 in a novel mouse model. Mice were viable and exhibited a normal lifespan. Initial studies did not reveal the presence of tumor development; however, altered nesting behavior and systemic lymphoid hyperplasia was noted in peripheral lymphoid organs. Alterations in T and B cell frequencies in the thymus and spleen were identified. Hence, exon skipping should be further investigated as a therapeutic approach for NF1 patients with pathogenic variants in exon 17, as homozygous deletion of exon 17 is consistent with at least partial function of neurofibromin.

3.
J Pers Med ; 11(12)2021 Dec 07.
Article in English | MEDLINE | ID: mdl-34945792

ABSTRACT

Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic disorder with almost 3000 different disease-causing variants within the NF1 gene identified. Up to 44% of these variants cause splicing errors to occur within pre-mRNA. A recurrent variant in exon 13, c.1466A>G; p.Y489C (Y489C) results in the creation of an intragenic cryptic splice site, aberrant splicing, a 62 base pair deletion from the mRNA, and subsequent frameshift. We investigated the ability of phosphorodiamidate morpholino oligomers (PMOs) to mask this variant on the RNA level, thus restoring normal splicing. To model this variant, we have developed a human iPS cell line homozygous for the variant using CRISPR/Cas9. PMOs were designed to be 25 base pairs long, and to cover the mutation site so it could not be read by splicing machinery. Results from our in vitro testing showed restoration of normal splicing in the RNA and restoration of full length neurofibromin protein. In addition, we observe the restoration of neurofibromin functionality through GTP-Ras and pERK/ERK testing. The results from this study demonstrate the ability of a PMO to correct splicing errors in NF1 variants at the RNA level, which could open the door for splicing corrections for other variants in this and a variety of diseases.

4.
Mol Ther Methods Clin Dev ; 20: 357-365, 2021 Mar 12.
Article in English | MEDLINE | ID: mdl-33553484

ABSTRACT

Lentiviral (LV) vectors based on human immunodeficiency virus type I (HIV-1) package two copies of their single-stranded RNA into vector particles. Normally, this RNA genome is reverse transcribed into a double-stranded DNA provirus that integrates into the cell genome, providing permanent gene transfer and long-term expression. Integration-deficient LV vectors have been developed to reduce the frequency of genomic integration and thereby limit their persistence in dividing cells. Here, we describe optimization of a reverse-transcriptase-deficient LV vector, which enables direct translation of LV RNA genomes upon cell entry, for transient expression of vector payloads as mRNA without a DNA intermediate. We have engineered a novel LV genome arrangement in which HIV-1 sequences are removed from the 5' end, to enable ribosomal entry from the 5' 7-methylguanylate cap for efficient translation of the vector payload. We have shown that this LV-mediated mRNA delivery platform provides transient transgene expression in vitro and in vivo. This has a potential application in gene and cell therapy scenarios requiring temporary payload expression in cells and tissues that can be targeted with pseudotyped LV vectors.

6.
J Biomed Mater Res B Appl Biomater ; 108(8): 3076-3083, 2020 11.
Article in English | MEDLINE | ID: mdl-32458591

ABSTRACT

There is a growing clinical demand in the wound care market to treat chronic wounds such as diabetic foot ulcers. Advanced cell and tissue-based products (CTPs) are often used to address challenging chronic wounds where healing has stalled. These products contain active biologics such as growth factors and cytokines as well as structural components that support and stimulate cell growth and assist in tissue regeneration. This study addresses the in vitro biologic effects of a clinically available dehydrated amniotic membrane allograft (DAMA). The broad mechanism of action results from DAMA's biologic composition that leads to stimulation of cell migration cell proliferation, and reduction of pro-inflammatory cytokines. Results show that DAMA possesses growth factors and cytokines such as EGF, FGF, PDGFs, VEGF, TGF-ß, IL-8, and TIMPs 1 and 2. Furthermore, in vitro experiments demonstrate that DAMA stimulates cell proliferation, cell migration, secretion of collagen type I, and the reduction of pro-inflammatory cytokines IL-1ß, IL-6, and TNF-α. This study findings are consistent with the clinical benefits previously published for DAMA and other CTPs in chronic wounds suggesting that the introduction of DAMA to non-healing, complex wounds helps to improve the wound milieu by providing essential structural components, cytokines, and growth factors to create an appropriate environment for wound healing.


Subject(s)
Amnion/transplantation , Biological Dressings , Wound Healing , Adult , Anti-Inflammatory Agents/pharmacology , Cell Adhesion , Cell Movement , Cell Proliferation , Cells, Cultured , Collagen Type I/metabolism , Cytokines/metabolism , Extracellular Matrix/drug effects , Female , Humans , Intercellular Signaling Peptides and Proteins/metabolism , Pregnancy
7.
J Tissue Eng Regen Med ; 14(3): 510-520, 2020 03.
Article in English | MEDLINE | ID: mdl-32012480

ABSTRACT

Recellularization of ex vivo-derived scaffolds remains a significant hurdle primarily due to the scaffolds subcellular pore size that restricts initial cell seeding to the scaffolds periphery and inhibits migration over time. With the aim to improve cell migration, repopulation, and graft mechanics, the effects of a four-step culture approach were assessed. Using an ex vivo-derived vein as a model scaffold, human smooth muscle cells were first seeded onto its ablumen (Step 1: 3 hr) and an aggressive 0-100% nutrient gradient (lumenal flow under hypotensive pressure) was created to initiate cell migration across the scaffold (Step 2: Day 0 to 19). The effects of a prolonged aggressive nutrient gradient created by this single lumenal flow was then compared with a dual flow (lumenal and ablumenal) in Step 3 (Day 20 to 30). Analyses showed that a single lumenal flow maintained for 30 days resulted in a higher proportion of cells migrating across the scaffold toward the vessel lumen (nutrient source), with improved distribution. In Step 4 (Day 31 to 45), the transition from hypotensive pressure (12/8 mmHg) to normotensive (arterial-like) pressure (120/80 mmHg) was assessed. It demonstrated that recellularized scaffolds exposed to arterial pressures have increased glycosaminoglycan deposition, physiological modulus, and Young's modulus. By using this stepwise conditioning, the challenging recellularization of a vein-based scaffold and its positive remodeling toward arterial biomechanics were obtained.


Subject(s)
Blood Vessel Prosthesis , Human Umbilical Vein Endothelial Cells/metabolism , Myocytes, Smooth Muscle/metabolism , Tissue Engineering , Tissue Scaffolds/chemistry , Biological Transport, Active , Cell Survival , Extracellular Matrix/chemistry , Humans
8.
J Pediatr Orthop ; 40(2): e122-e126, 2020 Feb.
Article in English | MEDLINE | ID: mdl-31045629

ABSTRACT

BACKGROUND: There are multiple methods of achieving upper extremity immobilization after pediatric elbow injuries; however, no biomechanical study has established an optimal construct. The goal of this study was to compare the strength of commonly used long arm splints and to evaluate the effect of reinforcing plaster splints with side struts. METHODS: Five categories of long arm posterior slab splints were tested: 4-inch plaster without side struts, 4-inch plaster with a medial side strut, 4-inch plaster with medial and lateral side struts, 5-inch plaster without side struts, and 4-inch fiberglass splint material without side struts. There were 4 splints in each group. As a control, 4 half fiberglass long arm casts were also tested. Each splint or cast was mounted on a single-column tensile tester and a 3-point bending load was applied to simulate an extension moment at the elbow. The maximum load before failure was measured and an ANOVA model was used to analyze the differences between groups. Additionally, a retrospective chart review was performed of pediatric patients who were immobilized postoperatively in a long arm plaster splint with side struts. We collected data on patient age, type of fracture, time from splint application in the operating room to removal in clinic, length of follow-up, and any complications. RESULTS: The 4-inch plaster splints reinforced with 2 struts had the highest average maximum load to failure (731±143 N), which was significantly higher than the 4-inch plaster splints with one strut (505±48 N) (P=0.01) and the 4-inch plaster splints without struts (100±10 N) (P<0.001). The half fiberglass casts failed at an average maximum load of 655±96 N, however there was no statistically significant difference compared with 4-inch plaster splints with 2 struts (P=0.10). The 5-inch plaster splints without side struts failed at a greater average maximum load (341±110 N) compared with the splints constructed with fiberglass material without side struts (233±61 N) (P=0.03). A total of 140 patients were identified in the retrospective review. Splint-related complications occurred in 2 patients. CONCLUSIONS: The addition of both 1 and 2 side struts to a 4-inch long arm plaster splint significantly increased the load to failure. The strength of 4-inch plaster splints with 2 side struts was comparable to that of half fiberglass casts. LEVEL OF EVIDENCE: NA (biomechanical study).


Subject(s)
Casts, Surgical , Splints , Biomechanical Phenomena , Child , Child, Preschool , Equipment Failure , Glass , Humans , Humeral Fractures/therapy , Male , Radius Fractures/therapy , Retrospective Studies , Splints/adverse effects , Tensile Strength , Elbow Injuries
9.
EMBO Mol Med ; 10(2): 254-275, 2018 02.
Article in English | MEDLINE | ID: mdl-29242210

ABSTRACT

Transferring large or multiple genes into primary human stem/progenitor cells is challenged by restrictions in vector capacity, and this hurdle limits the success of gene therapy. A paradigm is Duchenne muscular dystrophy (DMD), an incurable disorder caused by mutations in the largest human gene: dystrophin. The combination of large-capacity vectors, such as human artificial chromosomes (HACs), with stem/progenitor cells may overcome this limitation. We previously reported amelioration of the dystrophic phenotype in mice transplanted with murine muscle progenitors containing a HAC with the entire dystrophin locus (DYS-HAC). However, translation of this strategy to human muscle progenitors requires extension of their proliferative potential to withstand clonal cell expansion after HAC transfer. Here, we show that reversible cell immortalisation mediated by lentivirally delivered excisable hTERT and Bmi1 transgenes extended cell proliferation, enabling transfer of a novel DYS-HAC into DMD satellite cell-derived myoblasts and perivascular cell-derived mesoangioblasts. Genetically corrected cells maintained a stable karyotype, did not undergo tumorigenic transformation and retained their migration ability. Cells remained myogenic in vitro (spontaneously or upon MyoD induction) and engrafted murine skeletal muscle upon transplantation. Finally, we combined the aforementioned functions into a next-generation HAC capable of delivering reversible immortalisation, complete genetic correction, additional dystrophin expression, inducible differentiation and controllable cell death. This work establishes a novel platform for complex gene transfer into clinically relevant human muscle progenitors for DMD gene therapy.


Subject(s)
Chromosomes, Artificial, Human , Dystrophin/genetics , Genetic Therapy/methods , Muscular Dystrophy, Duchenne/genetics , Muscular Dystrophy, Duchenne/therapy , Animals , Cells, Cultured , Genetic Vectors , Humans , Mice , Models, Animal , Mutation
10.
Adv Healthc Mater ; 6(18)2017 Sep.
Article in English | MEDLINE | ID: mdl-28783879

ABSTRACT

Human perinatal tissues have been used for over a century as allogeneic biomaterials. Due to their advantageous properties including angiogenecity, anti-inflammation, anti-microbial, and immune privilege, these tissues are being utilized for novel applications across wide-ranging medical disciplines. Given continued clinical success, increased adoption of perinatal tissues as a disruptive technology platform has allowed for significant penetration into the multi-billion dollar biologics market. Here, we review current progress and future applications of perinatal biomaterials, as well as associated regulatory issues.


Subject(s)
Biocompatible Materials/chemistry , Humans , Regenerative Medicine/methods , Tissue Engineering/methods
11.
Drug Deliv Transl Res ; 6(2): 174-83, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26864696

ABSTRACT

A significant hurdle limiting musculoskeletal tissue regeneration is the inability to develop effective vascular networks to support cellular development within engineered constructs. Due to the inherent complexity of angiogenesis, where multiple biochemical pathways induce and control vessel formation, our laboratory has taken an alternate approach using a matrix material containing angiogenic and osteogenic proteins derived from human placental tissues. Single bolus administrations of the human placental matrix (hPM) have been shown to initiate angiogenesis but vascular networks deteriorated over time. Controlled/sustained delivery was therefore hypothesized to stabilize and extend network formation. To test this hypothesis, hPM was encapsulated in degradable poly(lactic-co-glycolic acid) (PLGA) microparticles to extend the release period. Microparticle preparation including loading, size, encapsulation efficiency, and release profile was optimized for hPM. The angiogenic cellular response to the hPM/PLGA-loaded microparticles was assessed in 3D alginate hydrogel matrices seeded with primary human endothelial cells. Results show an average microparticle diameter of 91.82 ± 2.92 µm, with an encapsulation efficiency of 75%, and a release profile extending over 30 days. Three-dimensional angiogenic assays with hPM-loaded PLGA microparticles showed initial stimulation of angiogenic tubules after 14 days and further defined network formations after 21 days of culture. Although additional optimization is necessary, these studies confirm the effectiveness of a novel controlled multi-protein release approach to induce and maintain capillary networks within alginate tissue scaffolds.


Subject(s)
Biocompatible Materials/pharmacology , Cell-Derived Microparticles/ultrastructure , Lactic Acid/pharmacokinetics , Neovascularization, Physiologic/drug effects , Placenta/chemistry , Polyglycolic Acid/pharmacokinetics , Biocompatible Materials/chemistry , Cell Culture Techniques , Cell-Derived Microparticles/chemistry , Cells, Cultured , Female , Human Umbilical Vein Endothelial Cells/drug effects , Humans , Lactic Acid/chemistry , Lactic Acid/pharmacology , Neovascularization, Pathologic , Particle Size , Polyglycolic Acid/chemistry , Polyglycolic Acid/pharmacology , Polylactic Acid-Polyglycolic Acid Copolymer , Pregnancy , Tissue Scaffolds
12.
Mol Ther ; 24(3): 465-74, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26765770

ABSTRACT

Programmable nucleases allow defined alterations in the genome with ease-of-use, efficiency, and specificity. Their availability has led to accurate and widespread genome engineering, with multiple applications in basic research, biotechnology, and therapy. With regard to human gene therapy, nuclease-based gene editing has facilitated development of a broad range of therapeutic strategies based on both nonhomologous end joining and homology-dependent repair. This review discusses current progress in nuclease-based therapeutic applications for a subset of inherited monogenic diseases including cystic fibrosis, Duchenne muscular dystrophy, diseases of the bone marrow, and hemophilia and highlights associated challenges and future prospects.


Subject(s)
Gene Editing , Genetic Diseases, Inborn/genetics , Genetic Diseases, Inborn/therapy , Genetic Therapy , Animals , Disease Models, Animal , Drug Evaluation, Preclinical , Gene Editing/methods , Gene Targeting , Gene Transfer Techniques , Genetic Therapy/methods , Humans , Translational Research, Biomedical
13.
Nucleic Acids Res ; 44(2): 744-60, 2016 Jan 29.
Article in English | MEDLINE | ID: mdl-26682797

ABSTRACT

Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disorder caused by the absence of dystrophin. We developed a novel gene therapy approach based on the use of the piggyBac (PB) transposon system to deliver the coding DNA sequence (CDS) of either full-length human dystrophin (DYS: 11.1 kb) or truncated microdystrophins (MD1: 3.6 kb; MD2: 4 kb). PB transposons encoding microdystrophins were transfected in C2C12 myoblasts, yielding 65±2% MD1 and 66±2% MD2 expression in differentiated multinucleated myotubes. A hyperactive PB (hyPB) transposase was then deployed to enable transposition of the large-size PB transposon (17 kb) encoding the full-length DYS and green fluorescence protein (GFP). Stable GFP expression attaining 78±3% could be achieved in the C2C12 myoblasts that had undergone transposition. Western blot analysis demonstrated expression of the full-length human DYS protein in myotubes. Subsequently, dystrophic mesoangioblasts from a Golden Retriever muscular dystrophy dog were transfected with the large-size PB transposon resulting in 50±5% GFP-expressing cells after stable transposition. This was consistent with correction of the differentiated dystrophic mesoangioblasts following expression of full-length human DYS. These results pave the way toward a novel non-viral gene therapy approach for DMD using PB transposons underscoring their potential to deliver large therapeutic genes.


Subject(s)
DNA Transposable Elements/genetics , Dystrophin/genetics , Genetic Therapy/methods , Muscular Dystrophy, Duchenne/pathology , Animals , Cell Differentiation , Cells, Cultured , Dogs , Dystrophin/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Male , Muscular Dystrophy, Duchenne/metabolism , Muscular Dystrophy, Duchenne/therapy , Myoblasts, Skeletal/cytology , Myoblasts, Skeletal/metabolism , Stem Cells/cytology , Stem Cells/metabolism , Transfection
14.
Biomaterials ; 49: 37-46, 2015 May.
Article in English | MEDLINE | ID: mdl-25725553

ABSTRACT

The inability to vascularize engineered organs and revascularize areas of infarction has been a major roadblock to delivering successful regenerative medicine therapies to the clinic. These investigations detail an isolated human extracellular matrix derived from the placenta (hPM) that induces vasculogenesis in vitro and angiogenesis in vivo within bioengineered tissues, with significant immune reductive properties. Compositional analysis showed ECM components (fibrinogen, laminin), angiogenic cytokines (angiogenin, FGF), and immune-related cytokines (annexins, DEFA1) in near physiological ratios. Gene expression profiles of endothelial cells seeded onto the matrix displayed upregulation of angiogenic genes (TGFB1, VEGFA), remodeling genes (MMP9, LAMA5) and vascular development genes (HAND2, LECT1). Angiogenic networks displayed a time dependent stability in comparison to current in vitro approaches that degrade rapidly. In vivo, matrix-dosed bioscaffolds showed enhanced angiogenesis and significantly reduced fibrosis in comparison to current angiogenic biomaterials. Implementation of this human placenta derived extracellular matrix provides an alternative to Matrigel and, due to its human derivation, its development may have significant clinical applications leading to advances in therapeutic angiogenesis techniques and tissue engineering.


Subject(s)
Extracellular Matrix/metabolism , Neovascularization, Physiologic , Animals , Capillaries/cytology , Capillaries/growth & development , Female , Fibrosis/pathology , Gene Expression Regulation , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Male , Pregnancy , Rats, Sprague-Dawley , Tissue Scaffolds/chemistry
15.
Tissue Eng Part A ; 19(17-18): 2005-13, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23541106

ABSTRACT

Whereas functionally different, both organogenesis and wound-healing processes create zones or regions of hypoxia that persist until capillary networks are formed to facilitate oxygen and nutrient delivery. Similarly, regenerative processes within in vitro engineered tissues experience the same hypoxic regions, but without the capacity to form functional capillaries resulting in a major limitation in developing full-thickness organs and tissues. Due to the importance of oxygen in wound healing and tissue regeneration, we hypothesize that directed oxygen gradients can be used to modulate cell function and promote more effective tissue regeneration. The effect of controlled oxygen gradients on human smooth muscle cells (SMCs) was assessed using dual chambered perfusion bioreactors to regulate transport conditions occurring in a model vascular construct. SMCs were seeded onto the ablumenal surface of the scaffold and cultured for 21 days under 3 independent gas environments: (1) 21% oxygen, (2) 11% oxygen, or (3) an ablumen to lumen oxygen gradient from 11% to 21%. When compared to 21% oxygen and 11% oxygen conditions, the directed 11%-21% oxygen gradient resulted in a raised metabolic activity and significantly improved cell migration. After 21 days from seeding, cells were shown to migrate entirely across the scaffold to the vessel lumen (>450 µm). Concomitant with a more uniform cell dispersion, scaffold mechanics were significantly enhanced with increased stiffness and tensile strength. Native oxygen gradients are known to play a pivotal role during organ development; these results show that directed oxygen gradients within in vitro systems can be used to facilitate early remodeling leading to significantly enhanced cell migration and scaffold biomechanics.


Subject(s)
Blood Vessel Prosthesis , Oxygen/chemistry , Tissue Engineering/methods , Bioreactors , Cell Movement/physiology , Cells, Cultured , Humans , Myocytes, Smooth Muscle/cytology , Tensile Strength , Tissue Scaffolds/chemistry
16.
J Biomed Mater Res A ; 100(8): 2194-203, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22623220

ABSTRACT

In vivo the vasculature provides an effective delivery system for cellular nutrients; however, artificial scaffolds have no such mechanism, and the ensuing limitations in mass transfer result in limited regeneration. In these investigations, the regional mass transfer properties that occur through a model scaffold derived from the human umbilical vein (HUV) were assessed. Our aim was to define the heterogeneous behavior associated with these regional variations, and to establish if different decellularization technologies can modulate transport conditions to improve microenvironmental conditions that enhance cell integration. The effect of three decellularization methods [Triton X-100 (TX100), sodium dodecyl sulfate (SDS), and acetone/ethanol (ACE/EtOH)] on mass transfer, cellular migration, proliferation, and metabolic activity were assessed. Results show that regional variation in tissue structure and composition significantly affects both mass transfer and cell function. ACE/EtOH decellularization was shown to increase albumin mass flux through the intima and proximate-medial region (0-250 µm) when compared with sections decellularized with TX100 or SDS; although, mass flux remained constant over all regions of the full tissue thickness when using TX100. Scaffolds decellularized with TX100 were shown to promote cell migration up to 146% further relative to SDS decellularized samples. These results show that depending on scaffold derivation and expectations for cellular integration, specificities of the decellularization chemistry affect the scaffold molecular architecture resulting in variable effects on mass transfer and cellular response.


Subject(s)
Tissue Engineering/methods , Tissue Scaffolds/chemistry , Animals , Biological Transport , Cattle , Cell Movement , Cell Proliferation , Diffusion , Dissection , Glucose/metabolism , Humans , Oxygen/metabolism , Potassium/metabolism , Serum Albumin, Bovine/metabolism , Umbilical Veins/cytology , Umbilical Veins/physiology
17.
Int J Pharm ; 360(1-2): 107-14, 2008 Aug 06.
Article in English | MEDLINE | ID: mdl-18565702

ABSTRACT

Injectable delivery systems are attractive as vehicles for localized delivery of therapeutics especially in the context of regenerative medicine. In this study, photocrosslinked polyanhydride (PA) networks were modified by incorporation of microparticles to modulate long-term delivery of macromolecules. The in vitro release of two model proteins (horseradish peroxidase (HRP) and bovine serum albumin labeled with fluorescein isothiocyanate (FITC-BSA)) were evaluated from networks composed of sebacic acid dimethacrylate (MSA), 1,6-bis-carboxyphenoxyhexane dimethacrylate (MCPH), poly(ethylene glycol) diacrylate (PEGDA), and calcium carbonate (CaCO3), supplemented with gelatin microparticles or sodium chloride crystals. Prior to incorporation into the networks, proteins were formulated into granules by dilution with a cyclodextrin excipient and gelatin-based wet-granulation. Protein release was modulated by incorporation of microparticles into photocrosslinked PA networks, presumably by enabling aqueous channels through the matrix. Furthermore, a dual release system has been demonstrated by incorporation of protein in both the PA matrix and the gelatin microparticles. These results suggest that microparticle incorporation into the photocrosslinked PA system may be a useful strategy to modulate protein release in injectable delivery systems for the long-term delivery of macromolecules. These composites present an interesting class of materials for bone regeneration applications.


Subject(s)
Proteins/administration & dosage , Cross-Linking Reagents , Fluorescein-5-isothiocyanate , Gelatin , Horseradish Peroxidase/administration & dosage , Horseradish Peroxidase/chemistry , Kinetics , Microscopy, Electron, Scanning , Nanoparticles , Particle Size , Photochemistry , Polymers , Proteins/chemistry , Proteins/pharmacokinetics , Serum Albumin, Bovine , Sodium Chloride , Solubility
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