Multifunctional Immunoliposomes Enhance the Immunotherapeutic Effects of PD-L1 Antibodies against Melanoma by Reprogramming Immunosuppressive Tumor Microenvironment.

The immunosuppressive tumor microenvironment (TME) can significantly limit the immunotherapeutic effects of the PD-L1 antibody (aPDL1) by inhibiting the infiltration of CD8+ cytotoxic T cells (CTLs) into the tumor tissues. However, how to reprogram the immunosuppressive TME and promote the infiltration of CTLs remains a huge challenge for aPDL1 to achieve the maximum benefits. Herein, the authors design a multifunctional immunoliposome that encapsulates the adrenergic receptor blocker carvedilol (CAR) and connects the "don't eat me" signal antibody (aCD47) and aPDL1 in series via a reactive oxygen species (ROS)-sensitive linker on the surface. In ROS-enriched immunosuppressive TME, the multifunctional immunoliposome (CAR@aCD47/aPDL1-SSL) can first release the outer aCD47 to block the "do not eat me" pathway, promote the phagocytosis of tumor cells by phagocytic cells, and activate CTLs. Then, the aPDL1 on the liposome surface is exposed to block the PD-1/PD-L1 signaling pathway, thereby inducing CTLs to kill tumor cells. CAR encapsulated in CAR@aCD47/aPDL1-SSL can block the adrenergic nerves in the tumor tissues and reduce their densities, thereby inhibiting angiogenesis in the tumor tissues and reprogramming the immunosuppressive TME. According to the results, CAR@aCD47/aPDL1-SSL holds an effective way to reprogram the immunosuppressive TME and significantly enhance immunotherapeutic efficiency of aPDL1 against the primary cancer and metastasis.

Tripeptide-based macroporous hydrogel improves the osteogenic microenvironment of stem cells.

Due to the ability to combine multiple osteogenic induction "cues" at the same time, hydrogels are widely used in the three-dimensional (3D) culture of human mesenchymal stem cells (hMSCs) and osteoinduction. However, the survival and proliferation of stem cells in a 3D culture system are limited, which reduces their osteogenic differentiation efficiency. In addition, the cells inside the hydrogel are prone to apoptosis due to hypoxia, which is a serious challenge for tissue engineering based on stem cells. In this study, a tripeptide-based macroporous alginate hydrogel was prepared to improve the osteogenic microenvironment of stem cells. The arginine-glycine-aspartate (RGD) peptide promoted the adhesion and proliferation of stem cells, and the degradation of gelatin microspheres (GMs) produced a macroporous structure to enhance further the migration and aggregation of stem cells. Mesoporous silica nanoparticles (MSNs) sustained-release bone-forming peptide-1 (BFP-1) induced osteogenic differentiation, and the sustained release of the QK peptide from the GMs promoted angiogenesis. In vitro experiments have shown that this functionalized hydrogel stimulates the proliferation of hMSCs, encourages larger cell cluster formation, and enhances the osteogenic differentiation efficiency. The released QK facilitates the proliferation and migration of endothelial cells. In vivo experiments have also verified that this system has a better osteogenic effect, and more blood vessels were observed inside the hydrogel, than in other systems. In general, this research has led to the development of a tripeptide macroporous hydrogel that can simultaneously promote osteogenesis and angiogenesis, showing great promise for applications of 3D cultures and stem cell-based tissue engineering.

A chondrogenesis induction system based on a functionalized hyaluronic acid hydrogel sequentially promoting hMSC proliferation, condensation, differentiation, and matrix deposition.

Hydrogel scaffolds are widely used in cartilage tissue engineering as a natural stem cell niche. In particular, hydrogels based on multiple biological signals can guide behaviors of mesenchymal stem cells (MSCs) during neo-chondrogenesis. In the first phase of this study, we showed that functionalized hydrogels with grafted arginine-glycine-aspartate (RGD) peptides and lower degree of crosslinking can promote the proliferation of human mesenchymal stem cells (hMSCs) and upregulate the expression of cell receptor proteins. Moreover, grafted RGD and histidine-alanine-valine (HAV) peptides in hydrogel scaffolds can regulate the adhesion of the intercellular at an early stage. In the second phase, we confirmed that simultaneous use of HAV and RGD peptides led to greater chondrogenic differentiation compared to the blank control and single-peptide groups. Furthermore, the controlled release of kartogenin (KGN) can better facilitate cell chondrogenesis compared to other groups. Interestingly, with longer culture time, cell condensation was clearly observed in the groups with RGD and HAV peptide. In all groups with RGD peptide, significant matrix deposition was observed, accompanied by glycosaminoglycan (GAG) and collagen (Coll) production. Through in vitro and in vivo experiments, this study confirmed that our hydrogel system can sequentially promote the proliferation, adhesion, condensation, chondrogenic differentiation of hMSCs, by mimicking the cell microenvironment during neo-chondrogenesis.

Injectable Porous Microchips with Oxygen Reservoirs and an Immune-Niche Enhance the Efficacy of CAR T Cell Therapy in Solid Tumors.

Chimeric antigen receptor (CAR) T cell therapy is a promising new class of hematological malignancy treatment. However, CAR T cells are rarely effective in solid tumor therapy mainly because of the poor trafficking of injected CAR T cells to the tumor site and their limited infiltration and survival in the immunosuppressive and hypoxic tumor microenvironment (TME). Here, we built an injectable immune-microchip (i-G/MC) system to intratumorally deliver CAR T cells and enhance their therapeutic efficacy in solid tumors. In the i-G/MC, oxygen carriers (Hemo) are released to disrupt the TME, and then, CAR T cells migrate from IL-15-laden i-G/MCs into the tumor stroma. The results indicate that Hemo and IL-15 synergistically enhanced CAR T cell survival and expansion under hypoxic conditions, promoting the potency and memory of CAR T cells. This i-G/MC not only serves as a cell carrier but also builds an immune-niche, enhancing the efficacy of CAR T cells.

Multifunctional Immunoliposomes Combining Catalase and PD-L1 Antibodies Overcome Tumor Hypoxia and Enhance Immunotherapeutic Effects Against Melanoma.

BACKGROUND: Immune checkpoint blockades (ICBs) are a promising treatment for cancers such as melanoma by blocking important inhibitory pathways that enable tumor cells to evade immune attack. Programmed death ligand 1 monoclonal antibodies (aPDL1s) can be used as an ICB to significantly enhance the effectiveness of tumor immunotherapy by blocking the PD-1/PD-L1 inhibitory pathway. However, the effectiveness of aPDL1s may be limited by low selectivity in vivo and immunosuppressed tumor microenvironment including hypoxia. PURPOSE: To overcome the limitations, we develop a multifunctional immunoliposome, called CAT@aPDL1-SSL, with catalase (CAT) encapsulated inside to overcome tumor hypoxia and aPDL1s modified on the surface to enhance immunotherapeutic effects against melanoma. METHODS: The multifunctional immunoliposomes (CAT@aPDL1-SSLs) are prepared using the film dispersion/post-insertion method. The efficacy of CAT@aPDL1-SSLs is verified by multiple experiments in vivo and in vitro. RESULTS: The results of this study suggest that the multifunctional immunoliposomes preserve and protect the enzyme activity of CAT and ameliorate tumor hypoxia. Moreover, the enhanced cellular uptake of CAT@aPDL1-SSLs in vitro and their in vivo biodistribution suggest that CAT@aPDL1-SSLs have great targeting ability,resulting in improved delivery and accumulation of immunoliposomes in tumor tissue.Finally, by activating and increasing the infiltration of CD8+ T cells at the tumor site, CAT@aPDL1-SSLs inhibit the growth of tumor and prolong survival time of mice,with low systemic toxicity. CONCLUSION: In conclusion, the multifunctional immunoliposomes developed and proposed in this study are a promising candidate for melanoma immunotherapy, and could potentially be combined with other cancer therapies like radiotherapy and chemotherapy to produce positive outcomes.

An effective dual-factor modified 3D-printed PCL scaffold for bone defect repair.

Numerous bioactive molecules produced in cells are involved in the process of bone formation. We consider that appropriate, simultaneous application of two types of bioactive molecules would accelerate the regeneration of tissues and organs. Therefore, we combined aspirin-loaded liposomes (Asp@Lipo) and bone forming peptide-1 (BFP-1) on three dimensional-printed polycaprolactone (PCL) scaffold and determined whether this system improved bone regeneration outcomes. in vitro experiments indicated that Asp@Lipo/BFP-1at a 3:7 ratio was the best option for enhancing the osteogenic efficiency of human mesenchymal stem cells (hMSCs). This was confirmed in an in vivo cranial defect animal model. In addition, RNA-Seq was applied for preliminarily exploration of the mechanism of action of this composite scaffold system, and the results suggested that it mainly improved bone regeneration via the PI3K/AKT signaling pathway. This approach will have potential for application in bone tissue engineering and regenerative medicine.

Triple-functional polyetheretherketone surface with enhanced bacteriostasis and anti-inflammatory and osseointegrative properties for implant application.

Polyetheretherketone (PEEK) is considered a potential orthopedic/dental material because of its excellent mechanical and chemical properties (e.g., similar elastic modulus to that of human bone). However, the poor bacteriostasis and anti-inflammatory and osseointegrative properties of bioinert PEEK impede its clinical application. We previously developed a facile and versatile surface modification method using dexamethasone plus minocycline-loaded liposomes (Dex/Mino liposomes) bonded by a mussel-inspired polydopamine coating, which effectively modulated cell inflammatory response and discouraged bacterial colonization in vitro. Herein, we report the application of this multifunctional surface modification method to improve bioinert PEEK, aimed at further studying the in vitro osteogenesis and in vivo properties of Dex/Mino liposome-modified PEEK to prevent bacterial contamination, attenuate the inflammatory response, and enhance ossification for physiologic osseointegration. Our study established that the Dex/Mino liposome-modified PEEK surface presented favorable stability and cytocompatibility. Compared with bare PEEK, improved osteogenic differentiation of human mesenchymal stem cells under both osteoinductive and osteoconductive conditions was found on the functionalized surface due to the liposomal Dex releasing. In vivo bacteriostasis assay confirmed that Mino released from the functionalized surface provided an effective antibacterial effect. Moreover, the subcutaneous foreign body reaction and beagle femur implantation models corroborated the enhanced anti-inflammatory and osteointegrative properties of the functionalized PEEK. Our findings indicate that the developed Dex/Mino liposome-modified PEEK with enhanced antibacterial, anti-inflammatory, and osseointegrative capacity has great potential as an orthopedic/dental implant material for clinical application.

A hybrid 3D-printed aspirin-laden liposome composite scaffold for bone tissue engineering.

Bone defects are some of the most difficult injuries to treat in clinical medicine. Evidence from cellular and animal studies suggests that aspirin exhibits protective effects on bone by promoting both the survival of osteoblast precursor stem cells and osteoblast differentiation. However, acquired resistance to aspirin and its cytotoxicity significantly limit its therapeutic application. Controlled release systems have been confirmed to promote the efficacy of certain drugs for bone regeneration. Additionally, the controlled release of a high dose of drug allows for lower dosing over an extended period. In this way, nano-liposomal encapsulation of aspirin can be used to reduce the cytotoxicity of the overall dose. Using a series of osteogenic experiments, this study found that an aspirin-laden liposome delivery system (Asp@Lipo) obviously promoted osteogenesis and immunomodulation of human mesenchymal stem cells (hMSCs). We also studied the in vitro capacity of polycaprolactone (PCL)-based bioactive composite (PCL-Asp@Lipo) scaffolds to facilitate cell proliferation and osteoblast differentiation. Compared to a common scaffold, ALP assays, immunofluorescence and calcium mineralisation studies revealed that the PCL-Asp@Lipo scaffolds enhanced the osteogenic differentiation of hMSCs. Subsequently, along with the cells, PCL and PCL-Asp@Lipo scaffolds were both implanted subcutaneously into nude mice for estimation of osteo-inductivity after 6 weeks, the PCL-Asp@Lipo composite scaffold exhibited more osteogenic activity than the bare PCL scaffold. This approach has potential applications in bone tissue repair and regenerative medicine.

Time-responsive osteogenic niche of stem cells: A sequentially triggered, dual-peptide loaded, alginate hybrid system for promoting cell activity and osteo-differentiation.

The efficacy of stem cell-based bone tissue engineering has been hampered by cell death and limited fate control. A smart cell culture system with the capability of sequentially delivering multiple factors in specific growth stages, like the mechanism of the natural extracellular matrix modulating tissue formation, is attractive for enhancing cell activity and controlling cell fate. Here, a bone forming peptide-1 (BFP-1)-laden mesoporous silica nanoparticles (pep@MSNs) incorporated adhesion peptide, containing the arginine-glycine-aspartic acid (RGD) domain, modified alginate hydrogel (RA) system (pep@MSNs-RA) was developed to promote the activity and stimulate osteo-differentiation of human mesenchymal stem cells (hMSCs) in sequence. The survivability and proliferation of hMSCs were enhanced in the adhesion peptide modified hydrogel. Next, BFP-1 released from pep@MSNs induced hMSCs osteo-differentiation after the proliferation stage. Moreover, BFP-1 near the cells was self-captured by the additional cell-peptide cross-linked networks formed by the ligands (RGD) binding to receptors on the cell surface, leading to long-term sustained osteo-stimulation of hMSCs. The results suggest that independent and sequential stimulation in proliferation and osteo-differentiation stages could synergistically enhance the survivability, expansion, and osteogenesis of hMSCs, as compared to stimulating alone or simultaneously. Overall, this study provided a new and valid strategy for stem cell expansion and osteo-differentiation in 2D or 3D culture systems, possessing potential applications in 3D bio-printing and tissue regeneration.