Extended Release of Bupivacaine from Temperature-responsive Hydrogels Provides Multi-day Analgesia for Postoperative Pain.

OBJECTIVE: A local anesthetic that provides analgesia lasting at least three days could significantly improve postoperative pain management. This study evaluated the analgesic efficacy and safety of an extended-release formulation of bupivacaine based on the injectable hydrogel carrier poly(N-isopropylacrylamide-co-dimethylbutyrolactone acrylamide-co-Jeffamine M-1000 acrylamide) (PNDJ). METHODS: The efficacy of PNDJ containing 4% bupivacaine (SBG004) given by peri-incisional subcutaneous injection (SBG004 SC) or wound filling instillation (SBG004 WF) was evaluated compared to saline, liposomal bupivacaine, bupivacaine collagen sponge, bupivacaine-meloxicam polyorthoester, and bupivacaine HCl in a porcine skin and muscle incision model. Mechanical allodynia was assessed by withdrawal from application of von Frey filaments, and local tolerance was evaluated by histology. Bupivacaine pharmacokinetics for SBG004 SC were measured in rabbits (16.5 mg bupivacaine/kg). RESULTS: Animals demonstrated less mechanical allodynia at incisions receiving SBG004 SC for up to 96 hours postoperatively. Incisions treated with SBG004 SC tolerated more force without a withdrawal indicative of pain compared to saline for 96 hours, and compared to SBG004 WF and all active controls at 24, 48, and 72 hours except bupivacaine-meloxicam polyorthoester at 72 hours. By 49 days, SBG004 was histologically absent and was replaced with granulation tissue infiltrated with immune cells in some areas. In rabbits, Cmax was 41.6 ± 9.7 ng/mL with t1/2 82.0 ± 35.8 hours (mean ± SD). CONCLUSIONS: Peri-incisional SBG004 SC provided extended release of bupivacaine sufficient to reduce sensation of incisional pain for 96 hours, in vivo bupivacaine delivery for at least 7 days, and a favorable local and systemic toxicity profile.

Local antimicrobial delivery from temperature-responsive hydrogels reduces incidence of intra-abdominal infection in rats.

The objective of this study was to evaluate local antimicrobial delivery from temperature-responsive hydrogels for preventing infection in a rat model of intra-abdominal infection (IAI), and to determine whether delivery of tobramycin and vancomycin in combination is effective against IAI pathogens. Rats received intraperitoneal inoculation of E. coli, rat cecal contents, or cecal contents supplemented with E. coli, and received either no treatment, subcutaneous cefoxitin, or local delivery from hydrogels containing vancomycin, tobramycin, or both antimicrobials. Only the hydrogel with tobramycin and vancomycin significantly increased the infection free-rate compared to no treatment for all inocula (E. coli: 13/17, p < 0.0001; cecal contents: 11/17, p = 0.0013; cecal contents + E. coli: 15/19, p < 0.0001). Additionally, tobramycin and vancomycin displayed no synergy or antagonism against clinical isolates in vitro. Local delivery of tobramycin and vancomycin from temperature-responsive hydrogels provides broad coverage and high antimicrobial concentrations for several hours that may be effective for preventing IAIs.

In situ crosslinking temperature-responsive hydrogels with improved delivery, swelling, and elasticity for endovascular embolization.

Endovascular embolization of cerebral aneurysms is a common approach for reducing the risk of often-fatal hemorrhage. However, currently available materials used to occlude these aneurysms provide incomplete filling (coils) or require a complicated, time-consuming delivery procedure (solvent-exchange precipitating polymers). The objective of this work was to develop an easily deliverable in situ forming hydrogel that can occlude the entire volume of an aneurysm. The hydrogel is formed by mixing a solution of a temperature-responsive polymer containing pendent thiol groups (poly(NIPAAm-co-cysteamine) or poly(NIPAAm-co-cysteamine-co-JAAm)) with a solution of poly(ethylene glycol) diacrylate (PEGDA). Incorporation of hydrophilic grafts of polyetheramine acrylamide (JAAm) in the temperature-responsive polymer caused weaker physical crosslinking, facilitated faster and more complete chemical crosslinking, and increased gel swelling. One formulation (30 wt % PNCJ20 + PEGDA) could be delivered for over 220 s after mixing, formed a strong and elastic hydrogel (G' > 6000 Pa) within 30 min and once set, maintained its shape and volume in a model aneurysm under flow. This gel represents a promising candidate water-based material utilizing both physical and chemical crosslinking that warrants further investigation as an embolic agent for saccular aneurysms.

In vivo evaluation of temperature-responsive antimicrobial-loaded PNIPAAm hydrogels for prevention of surgical site infection.

Surgical site infections (SSIs) are a persistent clinical challenge. Local antimicrobial delivery may reduce the risk of SSI by increasing drug concentrations and distribution in vulnerable surgical sites compared to what is achieved using systemic antimicrobial prophylaxis alone. In this work, we describe a comprehensive in vivo evaluation of the safety and efficacy of poly(N-isopropylacrylamide-co-dimethylbutyrolactone acrylamide-co-Jeffamine M-1000 acrylamide) [PNDJ], an injectable temperature-responsive hydrogel carrier for antimicrobial delivery in surgical sites. Biodistribution data indicate that PNDJ is primarily cleared via the liver and kidneys following drug delivery. Antimicrobial-loaded PNDJ was generally well-tolerated locally and systemically when applied in bone, muscle, articulating joints, and intraperitoneal space, although mild renal toxicity consistent with the released antimicrobials was identified at high doses in rats. Dosing of PNDJ at bone-implant interfaces did not affect normal tissue healing and function of orthopedic implants in a transcortical plug model in rabbits and in canine total hip arthroplasty. Finally, PNDJ was effective at preventing recurrence of implant-associated MSSA and MRSA osteomyelitis in rabbits, showing a trend toward outperforming commercially available antimicrobial-loaded bone cement and systemic antimicrobial administration. These studies indicate that antimicrobial-loaded PNDJ hydrogels are well-tolerated and could reduce incidence of SSI in a variety of surgical procedures.

Temperature-responsive PNDJ hydrogels provide high and sustained antimicrobial concentrations in surgical sites.

Local antimicrobial delivery is a promising strategy for improving treatment of deep surgical site infections (SSIs) by eradicating bacteria that remain in the wound or around its margins after surgical debridement. Eradication of biofilm bacteria can require sustained exposure to high antimicrobial concentrations (we estimate 100-1000 µg/mL sustained for 24 h) which are far in excess of what can be provided by systemic administration. We have previously reported the development of temperature-responsive hydrogels based on poly(N-isopropylacrylamide-co-dimethylbutyrolactone acrylate-co-Jeffamine M-1000 acrylamide) (PNDJ) that provide sustained antimicrobial release in vitro and are effective in treating a rabbit model of osteomyelitis when instilled after surgical debridement. In this work, we sought to measure in vivo antimicrobial release from PNDJ hydrogels and the antimicrobial concentrations provided in adjacent tissues. PNDJ hydrogels containing tobramycin and vancomycin were administered in four dosing sites in rabbits (intramedullary in the femoral canal, soft tissue defect in the quadriceps, intramuscular injection in the hamstrings, and intra-articular injection in the knee). Gel and tissue were collected up to 72 h after dosing and drug levels were analyzed. In vivo antimicrobial release (43-95% after 72 h) was markedly faster than in vitro release. Drug levels varied significantly depending on the dosing site but not between polymer formulations tested. Notably, total antimicrobial concentrations in adjacent tissue in all dosing sites were sustained at estimated biofilm-eradicating levels for at least 24 h (461-3161 µg/mL at 24 h). These results suggest that antimicrobial-loaded PNDJ hydrogels are promising for improving the treatment of biofilm-based SSIs.

In vitro susceptibility of Propionibacterium acnes to simulated intrawound vancomycin concentrations.

BACKGROUND: There is convincing evidence supporting the prophylactic use of intrawound vancomycin powder in spinal fusion surgery and mounting evidence in the arthroplasty literature suggesting that it can reduce surgical site infections. As a result, a number of shoulder arthroplasty surgeons have adopted this practice, despite a paucity of evidence and the presence of a pathogen that is, for the most part, unique to this area of the body-Propionibacterium acnes. The purpose of this study was to evaluate the efficacy of vancomycin against planktonic P. acnes in vitro, using time-dependent concentrations one would expect in vivo after intra-articular application. METHODS: Intrawound vancomycin concentrations were interpolated and extrapolated from existing in vivo data. Planktonic P. acnes was then subjected to a time-kill analysis during 96 hours. At each time point, the inoculum was centrifuged into pellet form and then reconstituted for serial drop counts onto blood agar plates. After anaerobic incubation, colony-forming units were counted, and log10 colony-forming units per milliliter were determined. RESULTS: Early time points grew to confluence, and thus colony-forming units per milliliter were not calculated. However, at 12 hours of vancomycin treatment, distinct colonies were appreciated. Notably, there was a 3 × log10 reduction in colony-forming units per milliliter between 12 and 48 hours, denoting bactericidal activity. In addition, P. acnes was completely eradicated after 3 days of treatment. CONCLUSION: When administered in a fashion meant to simulate time-dependent in vivo intrawound concentrations, vancomycin exhibited bactericidal activity against P. acnes. This may lend credence to the prophylactic use of vancomycin in shoulder surgery.

Temperature responsive hydrogels enable transient three-dimensional tumor cultures via rapid cell recovery.

Recovery of live cells from three-dimensional (3D) culture would improve analysis of cell behaviors in tissue engineered microenvironments. In this work, we developed a temperature responsive hydrogel to enable transient 3D culture of human glioblastoma (GBM) cells. N-isopropylacrylamide was copolymerized with hydrophilic grafts and functionalized with the cell adhesion peptide RGD to yield the novel copolymer poly(N-isopropylacrylamide-co-Jeffamine(®) M-1000 acrylamide-co-hydroxyethylmethacrylate-RGD), or PNJ-RGD. This copolymer reversibly gels in aqueous solutions when heated under normal cell culture conditions (37°C). Moreover, these gels redissolve within 70 s when cooled to room temperature without the addition of any agents to degrade the synthetic scaffold, thereby enabling rapid recollection of viable cells after 3D culture. We tested the efficiency of cell recovery following extended 3D culture and were able to recover more than 50% of viable GBM cells after up to 7 days in culture. These data demonstrate the utility of physically crosslinked PNJ-RGD hydrogels as a platform for culture and recollection of cells in 3D.

Bioengineered Scaffolds for 3D Analysis of Glioblastoma Proliferation and Invasion.

The invasion of malignant glioblastoma (GBM) cells into healthy brain is a primary cause of tumor recurrence and associated morbidity. Here, we describe a high-throughput method for quantitative measurement of GBM proliferation and invasion in three-dimensional (3D) culture. Optically clear hydrogels composed of thiolated hyaluronic acid and gelatin were chemically crosslinked with thiol-reactive poly(ethylene glycol) polymers to form an artificial 3D tumor microenvironment. Characterization of the viscoelasticity and aqueous stability indicated the hydrogels were mechanically tunable with stiffness ranging from 18 Pa to 18.2 kPa and were resistant to hydrolysis for at least 30 days. The proliferation, dissemination and subsequent invasion of U118 and U87R GBM spheroids cultured on the hydrogels were tracked in situ with repeated fluorescence confocal microscopy. Using custom automated image processing, cells were identified and quantified through 500 µm of gel over 14 days. Proliferative and invasive behaviors were observed to be contingent on cell type, gel stiffness, and hepatocyte growth factor availability. These measurements highlight the utility of this platform for performing quantitative, fluorescence imaging analysis of the behavior of malignant cells within an artificial, 3D tumor microenvironment.

In situ forming, resorbable graft copolymer hydrogels providing controlled drug release.

In situ forming hydrogels are promising drug delivery vehicles due to their ease of delivery as liquids and their ability to be used in sites with irregular geometries. In this work, we report on in situ forming, resorbable hydrogels based on N-isopropylacrylamide (NIPAAm) as a fluid-like controlled release gel. These gels are the first resorbable NIPAAm-based gels providing controlled release without relying on affinity between the drug and device. Therefore, these gels provide a more flexible delivery system which can be used to deliver any drug at a controlled rate. The polymers contain repeat units of NIPAAm with (R)-α-Acryloyloxy-ß,ß-dimethyl-γ-butyrolactone (DBLA) and varying amounts of hydrophilic Jeffamine® M-1000 acrylamide (JAAm) grafts. The graft copolymer architecture allows the water content of the hydrogels to be tuned over a wide range while keeping the initial gelation temperature below body temperature. Incorporation of JAAm in the polymers led to greater water content, faster gel degradation, and reduced burst release. Sustained release of the antimicrobial drugs cefazolin and vancomycin (over about 5 and 7 days, respectively) was observed from gels containing an intermediate amount of grafts which combined reduced phase separation with a degradation time of 40 days. The degradation byproducts of one hydrogel formulation were cytocompatible to NIH 3T3 fibroblasts at concentrations up to 2.5 wt %. This class of terpolymer hydrogels is a promising local delivery system for a wide variety of drugs, particularly for applications involving irregular geometries such as implant interfaces.

Bioresponsive copolymers of poly(N-isopropylacrylamide) with enzyme-dependent lower critical solution temperatures.

Novel thermoreversible copolymers of N-isopropylacrylamide (NIPAAm) with collagenase-sensitive solubility behavior were synthesized by radical polymerization of poly(NIPAAm-co-NASI) and nucleophilic substitution of custom peptides GAPGL-NH(2) and GAPGLF-NH(2). The materials were characterized by nuclear magnetic resonance spectroscopy (NMR), gel permeation chromatography in conjunction with static light scattering, differential scanning calorimetry (DSC), and cloud point determination. Successful synthesis and specific degradation by collagenase above and below the material LCST was confirmed by NMR. The LCST behavior of the polymers was affected by collagenase. The LCST of the copolymers, as measured by cloud point determination, increased by 1 and 9 degrees C, respectively, after enzymatic degradation. DSC thermographs indicated increased polymer solubility after enzymatic degradation because of a reduced energy of gelation. These results demonstrate the significant impact of a single amino acid on the LCST behavior of thermosensitive copolymers. Furthermore, the results suggest that comonomers in similar systems could be designed to elicit phase transitions or conformation changes in response to a variety of enzymes for which the substrate structure is known.