Lipid nanoparticle structure and delivery route during pregnancy dictate mRNA potency, immunogenicity, and maternal and fetal outcomes.

Treating pregnancy-related disorders is exceptionally challenging because the threat of maternal and/or fetal toxicity discourages the use of existing medications and hinders new drug development. One potential solution is the use of lipid nanoparticle (LNP) RNA therapies, given their proven efficacy, tolerability, and lack of fetal accumulation. Here, we describe LNPs for efficacious mRNA delivery to maternal organs in pregnant mice via several routes of administration. In the placenta, our lead LNP transfected trophoblasts, endothelial cells, and immune cells, with efficacy being structurally dependent on the ionizable lipid polyamine headgroup. Next, we show that LNP-induced maternal inflammatory responses affect mRNA expression in the maternal compartment and hinder neonatal development. Specifically, pro-inflammatory LNP structures and routes of administration curtailed efficacy in maternal lymphoid organs in an IL-1ß-dependent manner. Further, immunogenic LNPs provoked the infiltration of adaptive immune cells into the placenta and restricted pup growth after birth. Together, our results provide mechanism-based structural guidance on the design of potent LNPs for safe use during pregnancy.

Ionizable lipid nanoparticles deliver mRNA to pancreatic ß cells via macrophage-mediated gene transfer.

Systemic messenger RNA (mRNA) delivery to organs outside the liver, spleen, and lungs remains challenging. To overcome this issue, we hypothesized that altering nanoparticle chemistry and administration routes may enable mRNA-induced protein expression outside of the reticuloendothelial system. Here, we describe a strategy for delivering mRNA potently and specifically to the pancreas using lipid nanoparticles. Our results show that delivering lipid nanoparticles containing cationic helper lipids by intraperitoneal administration produces robust and specific protein expression in the pancreas. Most resultant protein expression occurred within insulin-producing ß cells. Last, we found that pancreatic mRNA delivery was dependent on horizontal gene transfer by peritoneal macrophage exosome secretion, an underappreciated mechanism that influences the delivery of mRNA lipid nanoparticles. We anticipate that this strategy will enable gene therapies for intractable pancreatic diseases such as diabetes and cancer.

The replacement of helper lipids with charged alternatives in lipid nanoparticles facilitates targeted mRNA delivery to the spleen and lungs.

The broad clinical application of mRNA therapeutics has been hampered by a lack of delivery vehicles that induce protein expression in extrahepatic organs and tissues. Recently, it was shown that mRNA delivery to the spleen or lungs is possible upon the addition of a charged lipid to a standard four-component lipid nanoparticle formulation. This approach, while effective, further complicates an already complex drug formulation and has the potential to slow regulatory approval and adversely impact manufacturing processes. We were thus motivated to maintain a four-component nanoparticle system while achieving shifts in tropism. To that end, we replaced the standard helper lipid in lipidoid nanoparticles, DOPE, with one of eight alternatives. These lipids included the neutral lipids, DOPC, sphingomyelin, and ceramide; the anionic lipids, phosphatidylserine (PS), phosphatidylglycerol, and phosphatidic acid; and the cationic lipids, DOTAP and ethyl phosphatidylcholine. While neutral helper lipids maintained protein expression in the liver, anionic and cationic lipids shifted protein expression to the spleen and lungs, respectively. For example, replacing DOPE with DOTAP increased positive LNP surface charge at pH 7 by 5-fold and altered the ratio of liver to lung protein expression from 36:1 to 1:56. Similarly, replacing DOPE with PS reduced positive charge by half and altered the ratio of liver to spleen protein expression from 8:1 to 1:3. Effects were consistent across ionizable lipidoid chemistries. Regarding mechanism, nanoparticles formulated with neutral and anionic helper lipids best transfected epithelial and immune cells, respectively. Further, the lung-tropic effect of DOTAP was linked to reduced immune cell infiltration of the lungs compared to neutral or anionic lipids. Together, these data show that intravenous non-hepatocellular mRNA delivery is readily achievable while maintaining a four-component formulation with modified helper lipid chemistry.

Inductive Remodeling of Extracellular Matrix Scaffolds in the Temporomandibular Joint of Pigs.

The temporomandibular joint (TMJ) disc is a fibrocartilaginous tissue located between the condyle of the mandible and glenoid fossa and articular eminence of the temporal bone. Damage or derangement of the TMJ disc can require surgical removal (discectomy) to restore function. Removal of the TMJ disc, however, leaves the joint space vulnerable to condylar remodeling and degradation, potentially leading to long-term complications. No consistently effective clinical option exists for repair or replacement of the disc following discectomy. This study investigates the use of an acellular scaffold composed of extracellular matrix (ECM) derived from small intestinal submucosa (SIS) as a regenerative template for the TMJ disc in a porcine model. Acellular SIS ECM scaffolds were implanted following discectomy and allowed to remodel for 2, 4, 12, and 24 weeks postimplantation. Remodeling of the implanted device was assessed by longitudinal magnetic resonance imaging (MRI) over the course of 6 months, as well as gross morphologic, histologic, biochemical, and biomechanical analysis (tension and compression) of explanted tissues (disc and condyle) at the time of sacrifice. When the scaffold remained in the joint space, longitudinal MRI demonstrated that the scaffolds promoted new tissue formation within the joint space throughout the study period. The scaffolds were rapidly populated with host-derived cells and remodeled with formation of new, dense, aligned fibrocartilage resembling native tissue as early as 1 month postimplantation. De-novo formation of peripheral muscular and tendinous attachments resembling those in native tissue was also observed. The remodeled scaffolds approached native disc biochemical composition and compressive modulus, and possessed 50% of the tensile modulus within 3 months postimplantation. No degradation of the condylar surface was observed. These results suggest that this acellular bioscaffold fills a medical need for which there is currently no effective treatment and may represent a clinically relevant "off-the-shelf" implant for reconstruction of the TMJ disc.

Distinct macrophage populations and phenotypes associated with IL-4 mediated immunomodulation at the host implant interface.

The host macrophage response to implants has shown to be affected by tissue location and physio-pathological conditions of the patient. Success in immunomodulatory strategies is thus predicated on the proper understanding of the macrophage populations participating on each one of these contexts. The present study uses an in vivo implantation model to analyze how immunomodulation via an IL-4 eluting implant affects distinct macrophage populations at the tissue-implant interface and how this may affect downstream regenerative processes. Populations identified as F4/80+, CD68+ and CD11b+ macrophages at the peri-implant space showed distinct susceptibility to polarize towards an M2-like phenotype under the effects of delivered IL-4. Also, the presence of the coating resulted in a significant reduction in F4/80+ macrophages, while other populations remained unchanged. These results suggests that the F4/80+ macrophage population may be predominant in the early stages of the host response at the surface of these implants, in contrast to CD11b+ macrophage populations which were either fewer in number or located more distant from the implant surface. Gene expression assays showed increased proteolytic activity and diminished matrix deposition as possible mechanisms explaining the decreased fibrotic capsule deposition and improved peri-implant tissue quality shown in previous studies using IL-4 eluting coatings. The pattern of M2-like gene expression promoted by IL-4 was correlated with glycosaminoglycan production within the site of implantation at early stages of the host response, suggesting a significant role in this response. These findings demonstrate that immunomodulatory strategies can be utilized to design and implement targeted delivery for improving biomaterial performance.

Inflammation and Ectopic Fat Deposition in the Aging Murine Liver Is Influenced by CCR2.

Aging is associated with inflammation and metabolic syndrome, which manifests in the liver as nonalcoholic fatty liver disease (NAFLD). NAFLD can range in severity from steatosis to fibrotic steatohepatitis and is a major cause of hepatic morbidity. However, the pathogenesis of NAFLD in naturally aged animals is unclear. Herein, we performed a comprehensive study of lipid content and inflammatory signature of livers in 19-month-old aged female mice. These animals exhibited increased body and liver weight, hepatic triglycerides, and inflammatory gene expression compared with 3-month-old young controls. The aged mice also had a significant increase in F4/80+ hepatic macrophages, which coexpressed CD11b, suggesting a circulating monocyte origin. A global knockout of the receptor for monocyte chemoattractant protein (CCR2) prevented excess steatosis and inflammation in aging livers but did not reduce the number of CD11b+ macrophages, suggesting changes in macrophage accumulation precede or are independent from chemokine (C-C motif) ligand-CCR2 signaling in the development of age-related NAFLD. RNA sequencing further elucidated complex changes in inflammatory and metabolic gene expression in the aging liver. In conclusion, we report a previously unknown accumulation of CD11b+ macrophages in aged livers with robust inflammatory and metabolic transcriptomic changes. A better understanding of the hallmarks of aging in the liver will be crucial in the development of preventive measures and treatments for end-stage liver disease in elderly patients.

Distinct release strategies are required to modulate macrophage phenotype in young versus aged animals.

The role of innate immunity and macrophages in the host response to biomaterials has received renewed attention. A context-dependent spectrum of macrophage phenotypes are shown to affect tissue integration and performance of implanted biomaterials and medical devices. Recent studies by our group demonstrated that the host response in aged animals was characterized by delayed macrophage recruitment, differences in marker expression and a shifted pro-inflammatory (M1) response, associated with an unresolved host response in the long-term. The present work sought to study the effects of single and sequential cytokine delivery regimens in aged mice to restore delayed recruitment of macrophages and shift the inflammatory host response towards an M2-like phenotype, using MCP-1 (macrophage chemotactic protein-1) and IL-4 (interleukin-4), respectively. Implantation of cytokine-eluting implants showed a preserved response to MCP-1 in both young and aged animals, restoring delayed macrophage recruitment in aged mice. However, the response elicited by IL-4, sequential delivery of MCP-1/IL-4 and coating components was distinct in young versus aged mice. While single delivery of IL-4 did not counteract the high inflammatory response observed in aged mice, the sequential delivery of MCP-1/IL-4 was capable of restoring both recruitment and shifting the macrophage response towards an M2-like phenotype, associated with decreased implant scarring in the long-term. In young mice, sequential delivery was not as effective as IL-4 alone at promoting an M2-like response, but did result in a reduction of M1 macrophages and capsule deposition downstream. These results demonstrate that a proper understanding of patient/context-dependent biological responses are needed to design biomaterial-based therapies with improved outcomes in the setting of aging.

Effect of Source Animal Age upon Macrophage Response to Extracellular Matrix Biomaterials.

Extracellular matrix biomaterials have been shown to promote constructive remodeling in many preclinical and clinical applications. This response has been associated with the promotion of a timely switch from pro-inflammatory (M1) to anti-inflammatory (M2) macrophages. A previous study has shown that this beneficial response is lost when these biomaterials are derived from aged animals. This study examined the impact of small intestine submucosa (SIS) derived from 12, 26 and 52 week old pigs on the phenotype and function of bone marrow macrophages derived either from 2 or 18 month old mice. Results showed that 52 week old SIS promoted less iNOS in 2 month macrophages and Fizz1 expression in 2 and 18 month compared to 12 week SIS. Pro-inflammatory cytokine exposure to 52 week SIS-treated macrophages resulted in higher iNOS in 18 month macrophages and reduced MHC-II expression in 2 month macrophages, as well as reduced nitric oxide production in comparison to 12 week SIS. These results indicate that ECM derived from aged animals promotes an altered macrophage phenotype compared to young controls. This suggests that sourcing of ECM from young donors is important to preserve constructive remodeling outcomes of ECM biomaterials. Alteration of macrophage phenotype by aged ECM also raises the hypothesis that alterations in aged ECM may play a role in immune dysfunction in aged individuals.

Injectable, porous, biohybrid hydrogels incorporating decellularized tissue components for soft tissue applications.

Biodegradable injectable hydrogels have been extensively studied and evaluated in various medical applications such as for bulking agents, drug delivery reservoirs, temporary barriers, adhesives, and cell delivery matrices. Where injectable hydrogels are intended to facilitate a healing response, it may be desirable to encourage rapid cellular infiltration into the hydrogel volume from the tissue surrounding the injection site. In this study, we developed a platform technique to rapidly form pores in a thermally responsive injectable hydrogel, poly(NIPAAm-co-VP-co-MAPLA) by using mannitol particles as porogens. In a rat hindlimb muscle injection model, hydrogels incorporating porosity had significantly accelerated cellular infiltration. To influence the inflammatory response to the injected hydrogel, enzymatically digested urinary bladder matrix (UBM) was mixed with the solubilized hydrogel. The presence of UBM was associated with greater polarization of the recruited macrophage population to the M2 phenotype, indicating a more constructive foreign body response. The hybrid hydrogel positively affected the wound healing outcomes of defects in rabbit adipose tissue with negligible inflammation and fibrosis, whereas scar formation and chronic inflammation were observed with autotransplantation and in saline injected groups. These results demonstrate the value of combining the effects of promoting cell infiltration and mediating the foreign body response for improved biomaterials options soft tissue defect filling applications. STATEMENT OF SIGNIFICANCE: Our objective was to develop a fabrication process to create porous injectable hydrogels incorporating decellularized tissue digest material. This new hydrogel material was expected to exhibit faster cellular infiltration and a greater extent of pro-M2 macrophage polarization compared to control groups not incorporating each of the functional components. Poly(NIPAAm-co-VP-co-MAPLA) was chosen as the representative thermoresponsive hydrogel, and mannitol particles and digested urinary bladder matrix (UBM) were selected as the porogen and the bioactive decellularized material components respectively. In rat hindlimb intramuscular injection models, this new hydrogel material induced more rapid cellular infiltration and a greater extent of M2 macrophage polarization compared to control groups not incorporating all of the functional components. The hybrid hydrogel positively affected the wound healing outcomes of defects in rabbit adipose tissue with negligible inflammation and fibrosis, whereas scar formation and chronic inflammation were observed with autotransplantation and in saline injected groups. The methodology of this report provides a straightforward and convenient mechanism to promote cell infiltration and mediate foreign body response in injectable hydrogels for soft tissue applications. We believe that the readership of Acta Biomaterialia will find the work of interest both for its specific results and general translatability of the findings.

Nerve-specific, xenogeneic extracellular matrix hydrogel promotes recovery following peripheral nerve injury.

Peripheral nerve possesses the inherent ability to regrow and recover following injury. However, nerve regeneration is often slow and incomplete due to limitations associated with the local microenvironment during the repair process. Manipulation of the local microenvironment at the site of nerve repair, therefore, represents a significant opportunity for improvement in downstream outcomes. Macrophages and Schwann cells play a key role in the orchestration of early events after peripheral nerve injury. We describe the production, characterization, and use of an injectable, peripheral nerve-specific extracellular matrix-based hydrogel (PNSECM) for promoting modulation of the local macrophage and Schwann cell responses at the site of nerve repair in a rodent model of sciatic nerve injury. We show that PNSECM hydrogels largely maintain the matrix structure associated with normal native peripheral nerve tissue. PNSECM hydrogels were also found to promote increased macrophage invasion, higher percentages of M2 macrophages and enhanced Schwann cell migration when used as a lumen filler in a rodent model of nerve gap repair using an inert nerve guidance conduit. These results suggest that an injectable PNSECM hydrogel can provide a supportive, bioactive scaffold which promotes repair of peripheral nerve in vivo. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 450-459, 2018.

Macrophage phenotype in response to ECM bioscaffolds.

Macrophage presence and phenotype are critical determinants of the healing response following injury. Downregulation of the pro-inflammatory macrophage phenotype has been associated with the therapeutic use of bioscaffolds composed of extracellular matrix (ECM), but phenotypic characterization of macrophages has typically been limited to small number of non-specific cell surface markers or expressed proteins. The present study determined the response of both primary murine bone marrow derived macrophages (BMDM) and a transformed human mononuclear cell line (THP-1 cells) to degradation products of two different, commonly used ECM bioscaffolds; urinary bladder matrix (UBM-ECM) and small intestinal submucosa (SIS-ECM). Quantified cell responses included gene expression, protein expression, commonly used cell surface markers, and functional assays. Results showed that the phenotype elicited by ECM exposure (MECM) is distinct from both the classically activated IFNγ+LPS phenotype and the alternatively activated IL-4 phenotype. Furthermore, the BMDM and THP-1 macrophages responded differently to identical stimuli, and UBM-ECM and SIS-ECM bioscaffolds induced similar, yet distinct phenotypic profiles. The results of this study not only characterized an MECM phenotype that has anti-inflammatory traits but also showed the risks and challenges of making conclusions about the role of macrophage mediated events without consideration of the source of macrophages and the limitations of individual cell markers.

Matrix-Bound Nanovesicles Recapitulate Extracellular Matrix Effects on Macrophage Phenotype.

The early macrophage response to biomaterials has been shown to be a critical and predictive determinant of downstream outcomes. When properly prepared, bioscaffolds composed of mammalian extracellular matrix (ECM) have been shown to promote a transition in macrophage behavior from a proinflammatory to a regulatory/anti-inflammatory phenotype, which in turn has been associated with constructive and functional tissue repair. The mechanism by which ECM bioscaffolds promote this phenotypic transition, however, is poorly understood. The present study shows that matrix-bound nanovesicles (MBV), a component of ECM bioscaffolds, are capable of recapitulating the macrophage activation effects of the ECM bioscaffold from which they are derived. MBV isolated from two different source tissues, porcine urinary bladder and small intestinal submucosa, were found to be enriched in miRNA125b-5p, 143-3p, and 145-5p. Inhibition of these miRNAs within macrophages was associated with a gene and protein expression profile more consistent with a proinflammatory rather than an anti-inflammatory/regulatory phenotype. MBV and their associated miRNA cargo appear to play a significant role in mediating the effects of ECM bioscaffolds on macrophage phenotype.

Effects of age-related shifts in cellular function and local microenvironment upon the innate immune response to implants.

The host macrophage response is now well recognized as a predictor of the success or failure of biomaterial implants following placement. More specifically, shifts from an "M1" pro-inflammatory towards a more "M2-like" anti-inflammatory macrophage polarization profile have been shown to result in enhanced material integration and/or tissue regeneration downstream. As a result, a number of biomaterials-based approaches to controlling macrophage polarization have been developed. However, the ability to promote such activity is predicated upon an in-depth, context-dependent understanding of the host response to biomaterials. Recent work has shown the impacts of both tissue location and tissue status (i.e. underlying pathology) upon the host innate immune response to implants, representing a departure from a focus upon implant material composition and form. Thus, the ideas of "biocompatibility," the host macrophage reaction, and ideal material requirements and modification strategies may need to be revisited on a patient, tissue, and disease basis. Immunosenescence, dysregulation of macrophage function, and delayed resolution of immune responses in aged individuals have all been demonstrated, suggesting that the host response to biomaterials in aged individuals should differ from that in younger individuals. However, despite the increasing usage of implantable medical devices in aged patients, few studies examining the effects of aging upon the host response to biomaterials and the implications of this response for long-term integration and function have been performed. The objective of the present manuscript is to review the putative effects of aging upon the host response to implanted materials and to advance the hypothesis that age-related changes in the local microenvrionement, with emphasis on the extracellular matrix, play a previously unrecognized role in determining the host response to implants.

Effects of aging upon the host response to implants.

Macrophage polarization during the host response is now a well-accepted predictor of outcomes following material implantation. Immunosenescence, dysregulation of macrophage function, and delayed resolution of immune responses in aged individuals have all been demonstrated, suggesting that host responses to materials in aged individuals should differ from those in younger individuals. However, few studies examining the effects of aging upon the host response have been performed. The present work sought to elucidate the impacts of aging upon the host response to polypropylene mesh implanted into 8-week-old and 18-month-old mice. The results showed that there are significant differences in macrophage surface marker expression, migration, and polarization during the early host macrophage response and delayed resolution of the host response in 18-month-old versus 8-week-old mice. These differences could not be attributed to cell-intrinsic defects alone, suggesting that the host macrophage response to implants is likely also dictated to a significant degree by the local tissue microenvironment. These results raise important questions about the design and testing of materials and devices often intended to treat aged individuals and suggest that an improved understanding of patient- and context-dependent macrophage responses has the potential to improve outcomes in aged individuals. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1281-1292, 2017.

Shifts in macrophage phenotype at the biomaterial interface via IL-4 eluting coatings are associated with improved implant integration.

The present study tests the hypothesis that transient, early-stage shifts in macrophage polarization at the tissue-implant interface from a pro-inflammatory (M1) to an anti-inflammatory/regulatory (M2) phenotype mitigates the host inflammatory reaction against a non-degradable polypropylene mesh material and improves implant integration downstream. To address this hypothesis, a nanometer-thickness coating capable of releasing IL-4 (an M2 polarizing cytokine) from an implant surface at early stages of the host response has been developed. Results of XPS, ATR-FTIR and Alcian blue staining confirmed the presence of a uniform, conformal coating consisting of chitosan and dermatan sulfate. Immunolabeling showed uniform loading of IL-4 throughout the surface of the implant. ELISA assays revealed that the amount and release time of IL-4 from coated implants were tunable based upon the number of coating bilayers and that release followed a power law dependence profile. In-vitro macrophage culture assays showed that implants coated with IL-4 promoted polarization to an M2 phenotype, demonstrating maintenance of IL-4 bioactivity following processing and sterilization. Finally, in-vivo studies showed that mice with IL-4 coated implants had increased percentages of M2 macrophages and decreased percentages of M1 macrophages at the tissue-implant interface during early stages of the host response. These changes were correlated with diminished formation of fibrotic capsule surrounding the implant and improved tissue integration downstream. The results of this study demonstrate a versatile cytokine delivery system for shifting early-stage macrophage polarization at the tissue-implant interface of a non-degradable material and suggest that modulation of the innate immune reaction at early stages of the host response may represent a preferred strategy for promoting biomaterial integration and success.

Macrophage polarization in response to ECM coated polypropylene mesh.

The host response to implanted biomaterials is a highly regulated process that influences device functionality and clinical outcome. Non-degradable biomaterials, such as knitted polypropylene mesh, frequently elicit a chronic foreign body reaction with resultant fibrosis. Previous studies have shown that an extracellular matrix (ECM) hydrogel coating of polypropylene mesh reduces the intensity of the foreign body reaction, though the mode of action is unknown. Macrophage participation plays a key role in the development of the foreign body reaction to biomaterials, and therefore the present study investigated macrophage polarization following mesh implantation. Spatiotemporal analysis of macrophage polarization was conducted in response to uncoated polypropylene mesh and mesh coated with hydrated and dry forms of ECM hydrogels derived from either dermis or urinary bladder. Pro-inflammatory M1 macrophages (CD86+/CD68+), alternatively activated M2 macrophages (CD206+/CD68+), and foreign body giant cells were quantified between 3 and 35 days. Uncoated polypropylene mesh elicited a dominant M1 response at the mesh fiber surface, which was decreased by each ECM coating type beginning at 7 days. The diminished M1 response was accompanied by a reduction in the number of foreign body giant cells at 14 and 35 days, though there was a minimal effect upon the number of M2 macrophages at any time. These results show that ECM coatings attenuate the M1 macrophage response and increase the M2/M1 ratio to polypropylene mesh in vivo.