Severe Acute Respiratory Syndrome Coronavirus-2 Spike Protein Nanogel as a Pro-Antigen Strategy with Enhanced Protective Immune Responses.

Prevention and intervention methods are urgently needed to curb the global pandemic of coronavirus disease-19 caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Herein, a general pro-antigen strategy for subunit vaccine development based on the reversibly formulated receptor binding domain of SARS-CoV-2 spike protein (S-RBD) is reported. Since the poor lymph node targeting and uptake of S-RBD by antigen-presenting cells prevent effective immune responses, S-RBD protein is formulated into a reversible nanogel (S-RBD-NG), which serves as a pro-antigen with enhanced lymph node targeting and dendritic cell and macrophage accumulation. Synchronized release of S-RBD monomers from the internalized S-RBD-NG pro-antigen triggers more potent immune responses in vivo. In addition, by optimizing the adjuvant used, the potency of S-RBD-NG is further improved, which may provide a generally applicable, safer, and more effective strategy for subunit vaccine development against SARS-CoV-2 as well as other viruses.

Liposomal Codelivery of Doxorubicin and Andrographolide Inhibits Breast Cancer Growth and Metastasis.

Effective treatment of metastatic (stage IV) breast cancers remains a formidable challenge. To address this issue, a cell-penetrating peptide-assisted liposomal system was developed for codelivery of doxorubicin and andrographolide. This nanomedicine-based combination therapy showed the ability to inhibit the in vitro migration and invasion of 4T1 cells through the wound healing and transwell invasion assays. Furthermore, this delivery system exhibited the enhanced accumulation in the tumor tissues and deep intratumoral penetration. The synergistic effect of doxorubicin and andrographolide led to an evident inhibition of tumor growth in an orthotopic breast tumor mouse model and efficient prevention of lung metastasis. The therapeutic mechanism was associated with the anti-angiogenesis effect. In conclusion, this nanomedicine-based combination therapy provides a potential method for overcoming metastatic breast cancers.

Reprogramming Tumor-Associated Macrophages To Reverse EGFRT790M Resistance by Dual-Targeting Codelivery of Gefitinib/Vorinostat.

Gefitinib is a first-line therapy in the EGFR-mutated nonsmall cell lung cancer (NSCLC). However, the development of drug resistance is almost unavoidable, thus leading to an unsustainable regimen. EGFRT790M mutation is the major cause responsible for the molecular-targeting therapy failure in NSCLC. Although the recently approved osimertinib is effective for the EGFRT790M-positive NSCLC, the osimertinib-resistant EGFR mutation is rapidly developed, too. In this study, we proposed a tumor-associated macrophage (TAM) reprogramming strategy for overcoming the EGFRT790M-associated drug resistance via a dual-targeting codelivery system of gefitinib/vorinostat that acted on both TAM with overexpression of mannose receptors and the HER-2 positive NSCLC cells. The trastuzumab-modified, mannosylated liposomal system was able to repolarize the protumor M2 phenotype to the antitumor M1 and cause the elevating ROS in the cancer cells, consequently modulating the intracellular redox balance via ROS/NOX3/MsrA axis. The suppressed MsrA facilitated the EGFRT790M degradation through 790M oxidation by ROS, thus resensitizing the EGFRT790M-positive cells to gefitinib. The dual-targeting codelivery and TAM-reprogramming strategies provided a potential method for rescuing the EGFRT790M-caused resistance to tyrosine kinase inhibitor treatment.

Codelivery of dihydroartemisinin and doxorubicin in mannosylated liposomes for drug-resistant colon cancer therapy.

Multidrug resistance (MDR) is a major hurdle in cancer chemotherapy and makes the treatment benefits unsustainable. Combination therapy is a commonly used method for overcoming MDR. In this study we investigated the anti-MDR effect of dihydroartemisinin (DHA), a derivative of artemisinin, in combination with doxorubicin (Dox) in drug-resistant human colon tumor HCT8/ADR cells. We developed a tumor-targeting codelivery system, in which the two drugs were co-encapsulated into the mannosylated liposomes (Man-liposomes). The Man-liposomes had a mean diameter of 158.8 nm and zeta potential of -15.8 mV. In the HCT8/ADR cells that overexpress the mannose receptors, the Man-liposomes altered the intracellular distribution of Dox, resulting in a high accumulation of Dox in the nuclei and thus displaying the highest cytotoxicity (IC50=0.073 µg/mL) among all the groups. In a subcutaneous HCT8/ADR tumor xenograft model, administration of the Man-liposomes resulted in a tumor inhibition rate of 88.59%, compared to that of 47.46% or 70.54%, respectively, for the treatment with free Dox or free Dox+DHA. The mechanisms underlying the anti-MDR effect of the Man-liposomes involved preferential nuclear accumulation of the therapeutic agents, enhanced cancer cell apoptosis, downregulation of Bcl-xl, and the induction of autophagy.

Nuclear-targeting TAT-PEG-Asp8-doxorubicin polymeric nanoassembly to overcome drug-resistant colon cancer.

AIM: Drug efflux-associated multidrug resistance (MDR) is a main obstacle to effective cancer chemotherapy. Large molecule drugs are not the substrates of P-glycoprotein, and can circumvent drug efflux and be retained inside cells. In this article we report a polymer-drug conjugate nanoparticulate system that can overcome MDR based on size-related exclusion effect. METHODS: Doxorubicin was coupled with the triblock polymeric material cell-penetrating TAT-PEG-poly(aspartic acid). The amphiphilic macromolecules (termed TAT-PEG-Asp8-Dox) could self-assemble into nanoparticles (NPs) in water. The antitumor activity was evaluated in drug-resistant human colon cancer HCT8/ADR cells in vitro and in nude mice bearing HCT8/ADR tumor. RESULTS: The self-assembling TAT-PEG-Asp8-Dox NPs were approximately 150 nm with a narrow particle size distribution, which not only increased the cellular uptake efficiency, but also bypassed P-glycoprotein-mediated drug efflux and improved the intracellular drug retention, thus yielding an enhanced efficacy for killing drug-resistant HCT8/ADR colon cancer cells in vitro. Importantly, the TAT-PEG-Asp8-Dox NPs enhanced the intranuclear disposition of drugs for grater inhibition of DNA/RNA biosynthesis. In nude mice bearing xenografted HCT8/ADR colon cancers, intravenous or peritumoral injection of TAT-PEG-Asp8-Dox NPs for 22 d effectively inhibited tumor growth. CONCLUSION: TAT-PEG-Asp8-Dox NPs can increase cellular drug uptake and intranuclear drug delivery and retain effective drug accumulation inside the cells, thus exhibiting enhanced anticancer activity toward the drug-resistant human colon cancer HCT8/ADR cells.

Weft-knitted silk-poly(lactide-co-glycolide) mesh scaffold combined with collagen matrix and seeded with mesenchymal stem cells for rabbit Achilles tendon repair.

Natural silk fibroin fiber scaffolds have excellent mechanical properties, but degrade slowly. In this study, we used poly(lactide-co-glycolide) (PLGA, 10:90) fibers to adjust the overall degradation rate of the scaffolds and filled them with collagen to reserve space for cell growth. Silk fibroin-PLGA (36:64) mesh scaffolds were prepared using weft-knitting, filled with type I collagen, and incubated with rabbit autologous bone marrow-derived mesenchymal stem cells (MSCs). These scaffold-cells composites were implanted into rabbit Achilles tendon defects. At 16 weeks after implantation, morphological and histological observations showed formation of tendon-like tissues that expressed type I collagen mRNA and a uniformly dense distribution of collagen fibers. The maximum load of the regenerated Achilles tendon was 58.32% of normal Achilles tendon, which was significantly higher than control group without MSCs. These findings suggest that it is feasible to construct tissue engineered tendon using weft-knitted silk fibroin-PLGA fiber mesh/collagen matrix seeded with MSCs for rabbit Achilles tendon defect repair.

Integrated platform of oxygen self-enriched nanovesicles: SP94 peptide-directed chemo/sonodynamic therapy for liver cancer.

Hepatocellular carcinoma (HCC) is a most common primary liver cancer among the most deadly malignancies. Selectively killing the cancer cells within the liver urgently requires the novel treatment strategies. The combination of sonodynamic therapy (SDT) and chemotherapy based on the nanotechnology have achieved some achievements in the HCC treatments. However, off-targeting drug delivery to healthy cells and the hypoxic microenvironment in the solid tumors frustrate the efforts to the combined strategy. The hypoxic microenvironment restrains the generation of ROS, leading to the decreased effects of SDT. To improve the clinical outcomes of chemo/SDT strategy, we created a novel oxygen self-enriched active targeted nanovesicle (ICG-DOX NPs/PFH@SP94-Lip). SP94 peptide could enhance the selectivity of the nanovesicles to liver tumor cells rather than normal liver cells. Besides, an oxygen carrier, perfluorohexanes (PFH), was co-loaded into liposomes to increase the oxygen level in tumor tissue, thus improving the effects of SDT. The in vivo studies showed that the ICG-DOX NPs/PFH@SP94-Lip combined with the external US stimulation significantly inhibited effects on tumor growth. Therefore, this novel oxygen self-enriched chemo/SDT nanocomposites represents a proof-of-concept liver tumor treatment strategy.

Effects of polyethylene glycol on the surface of nanoparticles for targeted drug delivery.

The rapid development of drug nanocarriers has benefited from the surface hydrophilic polymers of particles, which has improved the pharmacokinetics of the drugs. Polyethylene glycol (PEG) is a kind of polymeric material with unique hydrophilicity and electrical neutrality. PEG coating is a crucial factor to improve the biophysical and chemical properties of nanoparticles and is widely studied. Protein adherence and macrophage removal are effectively relieved due to the existence of PEG on the particles. This review discusses the PEGylation methods of nanoparticles and related techniques that have been used to detect the PEG coverage density and thickness on the surface of the nanoparticles in recent years. The molecular weight (MW) and coverage density of the PEG coating on the surface of nanoparticles are then described to explain the effects on the biophysical and chemical properties of nanoparticles.

Remodeling tumor immune microenvironment (TIME) for glioma therapy using multi-targeting liposomal codelivery.

BACKGROUND: Glioblastoma (GBM) treatment is undermined by the suppressive tumor immune microenvironment (TIME). Seek for effective methods for brain TIME modulation is a pressing need. However, there are two major challenges against achieving the goal: first, to screen the effective drugs with TIME-remodeling functions and, second, to develop a brain targeting system for delivering the drugs. METHODS: In this study, an α7 nicotinic acetylcholine receptors (nAChRs)-binding peptide DCDX was used to modify the codelivery liposomes to achieve a 'three-birds-one-stone' delivery strategy, that is, multi-targeting the glioma vessel endothelium, glioma cells, and tumor-associated macrophages that all overexpressed α7 nAChRs. A brain-targeted liposomal honokiol and disulfiram/copper codelivery system (CDX-LIPO) was developed for combination therapy via regulating mTOR (mammalian target of rapamycin) pathway for remodeling tumor metabolism and TIME. Honokiol can yield a synergistic effect with disulfiram/copper for anti-GBM. RESULTS: It was demonstrated that CDX-LIPO remarkably triggered tumor cell autophagy and induced immunogenic cell death, and meanwhile, activated the tumor-infiltrating macrophage and dendritic cells, and primed T and NK (natural killer) cells, resulting in antitumor immunity and tumor regression. Moreover, CDX-LIPO promoted M1-macrophage polarization and facilitated mTOR-mediated reprogramming of glucose metabolism in glioma. CONCLUSION: This study developed a potential combinatory therapeutic strategy by regulation of TIME and a 'three-birds-one-stone'-like glioma-targeting drug delivery system.

'Prodrug-Like' Acetylmannosamine Modified Liposomes Loaded With Arsenic Trioxide for the Treatment of Orthotopic Glioma in Mice.

Glioma is one of the fatal intracranial cancers that is a huge challenge to decrease the death rate currently. The deep penetration and high accumulation of therapeutic inorganic ions into the tumor site are extremely impeded due to the existence of physiological barriers, which limits to widen the indication of some drugs such as arsenic trioxide. The previous data have confirmed that the mannose substrate (MAN) without acetyl groups facilitates vesicles to go into the brain. Given that deacetylation of Ac4MAN groups on the surface of liposomes under the enzyme incubation occurred, namely 'prodrug-like' features of vesicles, the liposomes could more easily penetrate the BBB, target the glioma site, release arsenic trioxide, and inhibit the growth of glioma cells in the brain. Besides, the possibility of Ac4MAN binding to Gluts could be reduced due to the steric hindrance of acetyl groups, decreasing the off-target effects of vesicles. Here, we developed 'prodrug-like' arsenic trioxide (As2O3, ATO)-loaded liposomes inserted with distearoyl phospho-ethanolamine-polyethylene glycol-1000-p-carboxylpheny-α-d-acetylmannosamine (DSPE-PEG-1000-Ac4MAN), which was named Ac4MAN-ATO-LIP. Cytotoxic experiments of liposomes indicated that the toxicity of Ac4MAN-ATO-LIP was lower than that of free ATO but stronger than that of ATO-LIP (without insertion of DSPE-PEG-1000-Ac4MAN). The uptake of vesicles by U87 glioma cells displayed that the cellular uptake of Ac4MAN-Rho-LIP (labeled by rhodamine) was remarkably improved, compared with Rho-LIP. The in vivo biodistribution results showed the superiority of Ac4MAN-Rho-LIP in enhanced intracranial accumulation. Furthermore, the treatment of orthotopic glioma in Balb/c nude mice with Ac4MAN-ATO-LIP elongated the survival time of the animals than that with physiological saline, free ATO, or ATO-LIP, respectively. All the results suggested that the Ac4MAN-ATO-LIP had stronger anti-glioma effects as well as lower toxicities, and may be a promising approach for the treatment of brain cancer.

Targeting Regulation of Dually Modified Liposomes by Polyethylene Glycol Length of Vesicle Surface.

With aging of population, changing of living habits, and intake of high-fat diet, more and more people have been suffering from cardio-cerebral apoplexy. The synchronous treatment of cardio-cerebral conditions based on an integral strategy may bring benefit to the better clinical efficacy. The simultaneously-targeting delivery of active molecules by nanoscale carriers to heart and brain remains unmet problem. The physiological difference of targets between heart and brain makes it a huge challenge which one targeting ligand modification acquires the delivery of two organs and treatment, simultaneously. Traditionally, dually targeting strategies are introduced to enhance the selectivity for one aimed tissue and delivery efficiency of these particles. However, the interference between two targeting ligands on the surface of nanoscale carriers may influence the affinity of these ligands with their receptors or transporters, resulting to the change distribution of carriers. Herein, we observed that how anti-cardiac troponin I (cTnI) antibody (Ab) conjugated with the linker, polyethylene glycol (PEG), on the surface of liposomes influenced the affinity of mannose derivatives with transporter and regulated distribution of these vesicles in the heart and brain. The dually targeting liposomes can target to the heart and brain tissue simultaneously by the regulation length of PEG chain linking with p -pentanoic acid phenyl-α-D-acetylmannosamine (Ac4MAN). These results may bring benefit to design the multi-modification of nanocarriers and the treatment of cardio-cerebral diseases.

Nano-Structural Effects on Gene Transfection: Large, Botryoid-Shaped Nanoparticles Enhance DNA Delivery via Macropinocytosis and Effective Dissociation.

Effective delivery is the primary barrier against the clinical translation of gene therapy. Yet there remains too much unknown in the gene delivery mechanisms, even for the most investigated polymeric carrier (i.e., PEI). As a consequence, the conflicting results have been often seen in the literature due to the large variability in the experimental conditions and operations. Therefore, some key parameters should be identified and thus strictly controlled in the formulation process. Methods: The effect of the formulation processing parameters (e.g., concentration or mixture volume) and the resulting nanostructure properties on gene transfection have been rarely investigated. Two types of the PEI/DNA nanoparticles (NPs) were prepared in the same manner with the same dose but at different concentrations. The microstructure of the NPs and the transfection mechanisms were investigated through various microscopic methods. The therapeutic efficacy of the NPs was demonstrated in the cervical subcutaneous xenograft and peritoneal metastasis mouse models. Results: The high-concentration process (i.e., small reaction-volume) for mixture resulted in the large-sized PEI/DNA NPs that had a higher efficiency of gene transfection, compared to the small counterpart that was prepared at a low concentration. The microstructural experiments showed that the prepared small NPs were firmly condensed, whereas the large NPs were bulky and botryoid-shaped. The large NPs entered the tumor cells via the macropinocytosis pathway, and then efficiently dissociated in the cytoplasm and released DNA, thus promoting the intranuclear delivery. The enhanced in vivo therapeutic efficacy of the large NPs was demonstrated, indicating the promise for local-regional administration. Conclusion: This work provides better understanding of the effect of formulation process on nano-structural properties and gene transfection, laying a theoretical basis for rational design of the experimental process.