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We took a systems approach to the analysis of macrophage phenotype in regenerative and fibrotic volumetric muscle loss outcomes in mice together with analysis of systemic inflammation and of other leukocytes in the muscle, spleen, and bone marrow. Macrophage dysfunction in the fibrotic group occurred as early as day 1, persisted to at least day 28, and was associated with increased numbers of leukocytes in the muscle and bone marrow, increased pro-inflammatory marker expression in splenic macrophages, and changes in the levels of pro-inflammatory cytokines in the blood. The most prominent differences were in muscle neutrophils, which were much more abundant in fibrotic outcomes compared to regenerative outcomes at day 1 after injury. However, neutrophil depletion had little to no effect on macrophage phenotype or on muscle repair outcomes. Together, these results suggest that the entire system of immune cell interactions must be considered to improve muscle repair outcomes.

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Although most tissue types are capable of some form of self-repair and regeneration, injuries that are larger than a critical threshold or those occurring in the setting of certain diseases can lead to impaired healing and ultimately loss of structure and function. The immune system plays an important role in tissue repair and must be considered in the design of therapies in regenerative medicine. In particular, macrophage cell therapy has emerged as a promising strategy that leverages the reparative roles of these cells. Macrophages are critical for successful tissue repair and accomplish diverse functions throughout all phases of the process by dramatically shifting in phenotypes in response to microenvironmental cues. Depending on their response to various stimuli, they may release growth factors, support angiogenesis, and facilitate extracellular matrix remodeling. However, this ability to rapidly shift phenotype is also problematic for macrophage cell therapy strategies, because adoptively transferred macrophages fail to maintain therapeutic phenotypes following their administration to sites of injury or inflammation. Biomaterials are a potential way to control macrophage phenotype in situ while also enhancing their retention at sites of injury. Cell delivery systems incorporated with appropriately designed immunomodulatory signals have potential to achieve tissue regeneration in intractable injuries where traditional therapies have failed. Here we explorecurrent challenges in macrophage cell therapy, especially retention and phenotype control, how biomaterials may overcome them, and opportunities for next generation strategies. Biomaterials will be an essential tool to advance macrophage cell therapy for widespread clinical applications.

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Long-acting antiretroviral implants could help protect high-risk individuals from HIV infection. We describe the design and testing of a long-acting reservoir subcutaneous implant capable of releasing cabotegravir for several months. We compressed cabotegravir and excipients into cylindrical pellets and heat-sealed them in tubing composed of hydrophilic poly(ether-urethane) -. The implants have a 47 mm lumen length, 3.6 mm outer diameter, and 200 µm wall thickness. Four cabotegravir pellets were sealed in the membrane, with a total drug loading of 274 ± 3 mg. In vivo, the implants released 348 ± 107 µg/day (median value per implant, N = 41) of cabotegravir in rhesus macaques. Five implants generated an average cabotegravir plasma concentration of 373 ng/ml in rhesus macaques. The non-human primates tolerated the implant without gross pathology or microscopic signs of histopathology compared to placebo implants. Cabotegravir plasma levels in macaques dropped below detectable levels within two weeks after the removal of the implants.

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PURPOSE: Sexual transmission of HIV has been clinically proven to be preventable with a once-daily oral tablet; however, missed doses dramatically increase the risk of HIV infection. Long-acting subcutaneous implants do not allow the user to miss a dose. A desirable long-acting drug-eluting implant can deliver a constant amount of drug, adjust the delivered dose, and be readily manufactured. We present a long-acting, subcutaneous implant design composed of tenofovir alafenamide hemifumarate (TAF) pellets loaded in a sealed polyether urethane tube for the prevention of HIV transmission. METHODS: Implants were prepared with pressed drug pellets and extruded polyurethane tubing. In vitro release rate of implants using different pellet formulations, rate-controlling membranes, and geometries were measured. RESULTS: Tenofovir alafenamide release appeared to be governed by a pseudo-steady state and followed a mass transport model of release from a cylindrical drug reservoir. Implant seal integrity was tested and confirmed using mechanical testing. The inclusion of sodium chloride in the pellet increased the release rate and reduced initial lag. The release was sustained for 100 days. CONCLUSIONS: The release rate of tenofovir alafenamide mechanistically varied with geometry and rate controlling membrane composition. The polyether urethane implant presented herein is modular and tunable to adjust the release rate and duration of the TAF release.

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We describe the in vitro and in vivo evaluation of a subcutaneous reservoir implant delivering tenofovir alafenamide hemifumarate (TAF) for the prevention of HIV infection. These long-acting reservoir implants were able to deliver antiretroviral drug for over 90 days in vitro and in vivo We evaluated the implants for implantation site histopathology and pharmacokinetics in plasma and tissues for up to 12 weeks in New Zealand White rabbit and rhesus macaque models. A dose-ranging study in rabbits demonstrated dose-dependent pharmacokinetics and local inflammation up to severe necrosis around the active implants. The matched placebos showed normal wound healing and fibrous tissue encapsulation of the implant. We designed a second implant with a lower release rate and flux of TAF and achieved a median cellular level of tenofovir diphosphate of 42 fmol per 106 rhesus macaque peripheral blood mononuclear cells at a TAF dose of 10 µg/kg/day. This dose and flux of TAF also resulted in adverse local inflammation and necrosis near the implant in rhesus macaques. The level of inflammation in the primates was markedly lower in the placebo group than in the active-implant group. The histological inflammatory response to the TAF implant at 4 and 12 weeks in primates was graded as a severe reaction. Thus, while we were able to achieve a sustained target dose, we observed an unacceptable inflammatory response locally at the implant tissue interface.

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PURPOSE: Design of intravaginal rings (IVRs) for delivery of antiretrovirals is often guided by in vitro release under sink conditions, based on the assumption that in vivo release will follow a similar release profile. METHODS: We conducted a dose-ranging study in the female reproductive tract of pigtail macaques using matrix IVRs containing IQP-0528, a poorly soluble but highly potent antiretroviral drug with an IC90 of 146 ng/mL. These IVRs consisted of drug-loaded segments, 15.6% IQP-0528 in Tecoflex 85A, comprising either all, half, or a quarter of the entire ring. RESULTS: In vitro release under sink conditions demonstrates loading-proportional release, with a cumulative 30-day release of 48.5 ± 2.2 mg for our 100% loaded ring, 24.8 ± .36 mg from our 50% loaded ring, and 13.99 ± 1.58 mg from our 25% loaded ring. In vivo, while drug concentration in vaginal fluid is well in excess of IQP-0528's EC90, we find no statistical difference between the different ring loadings in either swab drug levels or drug released from our rings. CONCLUSIONS: We show that in vitro release may not accurately reflect in vivo release, particularly for poorly soluble drugs. All tested loadings of our IVRs are capable of delivering IQP-0528 well in excess of the IC90.

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BACKGROUND: Reported vaginal and seminal fluid simulants have complex compositions with multiple preparatory steps that contribute to physical instability. We report the design and characterization of stable and simplified buffers that mimic the salient physical/chemical properties of the physiological fluids. STUDY DESIGN/METHODS: Human cervicovaginal and seminal fluid samples were collected and buffering capacity was determined. The major buffering species were identified from published compositions of reproductive tract fluids. These values were used to compute the composition of vaginal and seminal fluid simulants. Ionic strength, buffering capacities, pH and osmolalities were then calculated or experimentally determined. Finally, cytotoxicity was evaluated in HEC-1-A cells and 3D reconstructed EpiVaginal™ tissue (VEC-100-FT) using naïve cells/tissue and nonoxynol-9 as controls. RESULTS: The use of calculated amounts of conjugate acid and base for buffer development resulted in compositions that did not require endpoint pH adjustment and could be formulated as stable 10× concentrates. Furthermore, due to the absence of complex divalent salts, all our proposed simulants were stable at 4 °C for 1 month whereas precipitation and pH and osmolality changes were noted in reported buffers. Experimental determination of buffering capacities yielded similar values for undiluted cervicovaginal fluid (ß4.2-5.2=35.6 ± 12.3 mM, N=7) and human seminal fluid (ß7-6=37.5 ± 5 mM, N=3). All neat simulants showed significant cytotoxicity in HEC-1-A cells but were well tolerated by organotypic vaginal tissue. CONCLUSIONS: We report revised and improved compositions of buffers mimicking salient properties of vaginal and seminal fluid necessary for in vitro product evaluation. IMPLICATIONS: To support research in reproductive health and in particular drug delivery, we have designed and characterized stable new media to mimic these important fluids that can be used in a variety of in vitro studies.

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