mRNA-Laden Lipid-Nanoparticle-Enabled in Situ CAR-Macrophage Engineering for the Eradication of Multidrug-Resistant Bacteria in a Sepsis Mouse Model.

Sepsis, which is the most severe clinical manifestation of acute infection and has a mortality rate higher than that of cancer, represents a significant global public health burden. Persistent methicillin-resistant Staphylococcus aureus (MRSA) infection and further host immune paralysis are the leading causes of sepsis-associated death, but limited clinical interventions that target sepsis have failed to effectively restore immune homeostasis to enable complete eradication of MRSA. To restimulate anti-MRSA innate immunity, we developed CRV peptide-modified lipid nanoparticles (CRV/LNP-RNAs) for transient in situ programming of macrophages (MΦs). The CRV/LNP-RNAs enabled the delivery of MRSA-targeted chimeric antigen receptor (CAR) mRNA (SasA-CAR mRNA) and CASP11 (a key MRSA intracellular evasion target) siRNA to MΦs in situ, yielding CAR-MΦs with boosted bactericidal potency. Specifically, our results demonstrated that the engineered MΦs could efficiently phagocytose and digest MRSA intracellularly, preventing immune evasion by the "superbug" MRSA. Our findings highlight the potential of nanoparticle-enabled in vivo generation of CAR-MΦs as a therapeutic platform for multidrug-resistant (MDR) bacterial infections and should be confirmed in clinical trials.

Surficial nano-deposition locoregionally yielding bactericidal super CAR-macrophages expedites periprosthetic osseointegration.

Tracking and eradicating Staphylococcus aureus in the periprosthetic microenvironment are critical for preventing periprosthetic joint infection (PJI), yet effective strategies remain elusive. Here, we report an implant nanoparticle coating that locoregionally yields bactericidal super chimeric antigen receptor macrophages (CAR-MΦs) to prevent PJI. We demonstrate that the plasmid-laden nanoparticle from the coating can introduce S. aureus-targeted CAR genes and caspase-11 short hairpin RNA (CASP11 shRNA) into macrophage nuclei to generate super CAR-MΦs in mouse models. CASP11 shRNA allowed mitochondria to be recruited around phagosomes containing phagocytosed bacteria to deliver mitochondria-generated bactericidal reactive oxygen species. These super CAR-MΦs targeted and eradicated S. aureus and conferred robust bactericidal immunologic activity at the bone-implant interface. Furthermore, the coating biodegradability precisely matched the bone regeneration process, achieving satisfactory osteogenesis. Overall, our work establishes a locoregional treatment strategy for priming macrophage-specific bactericidal immunity with broad application in patients suffering from multidrug-resistant bacterial infection.

Mussel-inspired alkaline phosphatase-specific coating on orthopedic implants for spatiotemporal modulating local osteoimmune microenvironment to facilitate osseointegration.

Inadequate initial osseointegration and consequent prosthesis loosening are the most severe complications after artificial arthroplasty. Proper immune responses are crucial for the successful implantation of artificial prostheses. Macrophages are central in osteoimmunomodulation because they exert distinct functions with highly plasticity. Herein, we developed an alkaline phosphatase (ALP) sensitive mussel-inspired coating on orthopedic implants for promoting osseointegration. First, the resveratrol-alendronate complexes were deposited on titanium implant surface through mussel-inspired interfacial interactions. Upon prosthesis implantation, macrophages first polarized towards M1 type to initiate inflammatory responses and bone regeneration. As osteogenesis progresses, increasing amounts of ALP secreted by osteoblasts was cleaved the resveratrol-alendronate complexes. Then, the released resveratrol further promoted osteogenic differentiation of BMSCs and induced locoregional macrophages M2 polarization. Our results demonstrated that the bioinspired osteoimmunomodulation coating remarkably facilitated the prosthesis-bone integration by spatiotemporally modulating macrophages switching from M1 to M2 polarization in response to a real-time healing signal during osteogenesis. In summary, the mussel-inspired osteoimmunomodulation coating technology may provide a new approach for promoting osseointegration after artificial arthroplasty.

Remodeling articular immune homeostasis with an efferocytosis-informed nanoimitator mitigates rheumatoid arthritis in mice.

Massive intra-articular infiltration of proinflammatory macrophages is a prominent feature of rheumatoid arthritis (RA) lesions, which are thought to underlie articular immune dysfunction, severe synovitis and ultimately joint erosion. Here we report an efferocytosis-informed nanoimitator (EINI) for in situ targeted reprogramming of synovial inflammatory macrophages (SIMs) that thwarts their autoimmune attack and reestablishes articular immune homeostasis, which mitigates RA. The EINI consists of a drug-based core with an oxidative stress-responsive phosphatidylserine (PtdSer) corona and a shell composed of a P-selectin-blocking motif, low molecular weight heparin (LMWH). When systemically administered, the LMWH on the EINI first binds to P-selectin overexpressed on the endothelium in subsynovial capillaries, which functions as an antagonist, disrupting neutrophil synovial trafficking. Due to the strong dysregulation of the synovial microvasculature, the EINI is subsequently enriched in the joint synovium where the shell is disassembled upon the reactive oxygen species stimulation, and PtdSer corona is then exposed. In an efferocytosis-like manner, the PtdSer-coroneted core is in turn phagocytosed by SIMs, which synergistically terminate SIM-initiated pathological cascades and serially reestablish intra-articular immune homeostasis, conferring a chondroprotective effect. These findings demonstrate that SIMs can be precisely remodeled via the efferocytosis-mimetic strategy, which holds potential for RA treatment.

Exosome-Functionalized Ti6Al4V Scaffolds Promoting Osseointegration by Modulating Endogenous Osteogenesis and Osteoimmunity.

Periprosthetic bone defects are the most serious problem of revision total hip arthroplasty, which can easily lead to insufficient osteointegration between the prosthesis and host bone. Bone marrow mesenchymal stem cells (BMSCs) and a moderate inflammatory response at the prosthesis-bone interface play an important role in osteointegration. Here, we developed microarc oxide titanium implant loaded engineered exosomes (S-Exos) to promote osseointegration at the prosthesis-bone interface. First, Smurf1-shRNA was transferred into the BMSCs using a viral vector to prepare S-Exos, which were subsequently immobilized to the microarc oxide titanium implant surface with positively charged polyethyleneimine. The immobilized S-Exos could be slowly and uniformly released and subsequently phagocytosed by BMSCs and macrophages. Once the S-Exos were phagocytosed, they could simultaneously activate the BMP/Smad signaling pathway in the BMSCs and promote macrophage M2 polarization, both of which enhance osseointegration. Specifically, this S-Exos coating exhibits a dual effect of promoting osseointegration, including the osseointegration of BMSCs by activating the BMP/Smad signaling pathway and the macrophage M2 polarization promoting osseointegration. In summary, the construction of S-Exos modified microarc oxide titanium implants could provide a new method for promoting osteointegration between the prosthesis and host bone in revision total hip arthroplasty.

Simultaneously promoting adhesion and osteogenic differentiation of bone marrow-derived mesenchymal cells by a functional electrospun scaffold.

Electrospinning is a common technology to construct tissue engineering scaffolds for bone regeneration. However, pure electrospun scaffolds do not enrich seed cells or promote their osteogenic differentiation. Biological functionalization of tissue engineering scaffolds is currently a hot research topic. Therefore, in this study, the bone marrow-derived mesenchymal cells (BM-MSC)-specific affinity peptide E7 and a bone morphogenic protein 2 (BMP-2) mimetic peptide were concomitantly conjugated onto the surface of an electrospun scaffold to construct a functional PEB scaffold. Characterization of PEB scaffolds revealed that both E7 and BMP-2 mimetic peptides were successfully conjugated onto the surface of electrospun scaffolds. With regard to biological activity, the PEB scaffold could synchronously promote adhesion and osteogenic differentiation of BM-MSC as a result of the co-delivery of E7 and BMP-2 mimetic peptides, which proved superior compared with the other three scaffolds. Consequently, the PEB scaffold offers a new concept for the construction of bone tissue engineering scaffolds.