Copper Deposition in Polydopamine Nanostructure to Promote Cuproptosis by Catalytically Inhibiting Copper Exporters of Tumor Cells for Cancer Immunotherapy.

Cuproptosis is an emerging programmed cell death, displaying great potential in cancer treatment. However, intracellular copper content to induce cuproptosis is unmet, which mainly ascribes to the intracellular pumping out equilibrium mechanism by copper exporter ATP7A and ATP7B. Therefore, it is necessary to break such export balance mechanisms for desired cuproptosis. Mediated by diethyldithiocarbamate (DTC) coordination, herein a strategy to efficiently assemble copper ions into polydopamine nanostructure (PDA-DTC/Cu) for reprogramming copper metabolism of tumor is developed. The deposited Cu2+ can effectively trigger the aggregation of lipoylated proteins to induce cuproptosis of tumor cells. Beyond elevating intracellular copper accumulation, PDA-DTC/Cu enables to break the balance of copper metabolism by disrupting mitochondrial function and restricting the adenosine triphosphate (ATP) energy supply, thus catalytically inhibiting the expressions of ATP7A and ATP7B of tumor cells to enhance cuproptosis. Meanwhile, the killed tumor cells can induce immunogenic cell death (ICD) to stimulate the immune response. Besides, PDA-DTC/Cu NPs can promote the repolarization of tumor-associated macrophages (TAMs ) to relieve the tumor immunosuppressive microenvironment (TIME). Collectively, PDA-DTC/Cu presented a promising "one stone two birds" strategy to realize copper accumulation and inhibit copper export simultaneously to enhance cuproptosis for 4T1 murine breast cancer immunotherapy.

Nanofactory for metabolic and chemodynamic therapy: pro-tumor lactate trapping and anti-tumor ROS transition.

Lactate plays a critical role in tumorigenesis, invasion and metastasis. Exhausting lactate in tumors holds great promise for the reversal of the immunosuppressive tumor microenvironment (TME). Herein, we report on a "lactate treatment plant" (i.e., nanofactory) that can dynamically trap pro-tumor lactate and in situ transformation into anti-tumor cytotoxic reactive oxygen species (ROS) for a synergistic chemodynamic and metabolic therapy. To this end, lactate oxidase (LOX) was nano-packaged by cationic polyethyleneimine (PEI), assisted by a necessary amount of copper ions (PLNPCu). As a reservoir of LOX, the tailored system can actively trap lactate through the cationic PEI component to promote lactate degradation by two-fold efficiency. More importantly, the byproducts of lactate degradation, hydrogen peroxide (H2O2), can be transformed into anti-tumor ROS catalyzing by copper ions, mediating an immunogenic cell death (ICD). With the remission of immunosuppressive TME, ICD process effectively initiated the positive immune response in 4T1 tumor model (88% tumor inhibition). This work provides a novel strategy that rationally integrates metabolic therapy and chemodynamic therapy (CDT) for combating tumors.

Engineering of a Pluronic F127 functionalized magnetite/graphene nanohybrid for chemophototherapy.

In this study, a multifunctional graphene based nanohybrid, termed as GN/Fe3O4/PF127, is engineered via a facile one-pot process consisting of simultaneous reduction of graphene oxide/Fe3O4 and subsequent assembly of Pluronic F127 (PF127) onto graphene nanosheets (GNs). The unique aromatic and planar structure of GNs allows the attachment of multiple functional components including MRI contrast agent (Fe3O4 nanoparticles) and an aromatic anticancer drug like doxorubicin (DOX), as well as PF127 coating which imparts physiological dispersivity and stability to the nanohybrid. The successful assembly process is revealed by TEM observation, size and FITR monitoring. In contrast with the primitive graphene or its oxide derivative, the resulting GN/Fe3O4/PF127 nanohybrids have shown high biological dispersion and MRI effect for diagnosis due to the incorporation of superparamagnetic Fe3O4 nanoparticles without evident cytotoxicity. Moreover, the GN/Fe3O4/PF127 nanohybrid exhibits a photothermal effect due to the considerable optical absorption in the near-infrared region of GNs. The GN/Fe3O4/PF127 nanohybrid could be a further platform for chemophototherapy assisted by the therapeutic DOX. Cellular toxicity assays indicated that the DOX-loaded GN/Fe3O4/PF127 nanohybrid showed a remarkable cytotoxicity to HeLa cells and the cytotoxic effect was intensified when subjected to photoirradiation. Confocal laser scanning microscopy (CLSM) and flow cytometric analysis (FCAS) revealed that the nanohybrid could be easily uptaken into HeLa cells.

Engineered redox-responsive PEG detachment mechanism in PEGylated nano-graphene oxide for intracellular drug delivery.

In biomedical applications, polyethylene glycol (PEG) functionalization has been a major approach to modify nanocarriers such as nano-graphene oxide for particular biological requirements. However, incorporation of a PEG shell poses a significant diffusion barrier that adversely affects the release of the loaded drugs. This study addresses this critical issue by employing a redox-responsive PEG detachment mechanism. A PEGylated nano-graphene oxide (NGO-SS-mPEG) with redox-responsive detachable PEG shell is developed that can rapidly release an encapsulated payload at tumor-relevant glutathione (GSH) levels. The PEG shell grafted onto NGO sheets gives the nanocomposite high physiological solubility and stability in circulation. It can selectively detach from NGO upon intracellular GSH stimulation. The surface-engineered structures are shown to accelerate the release of doxorubicin hydrochloride (DXR) from NGO-SS-mPEG 1.55 times faster than in the absence of GSH. Confocal microscopy shows clear evidence of NGO-SS-mPEG endocytosis in HeLa cells, mainly accumulated in cytoplasm. Furthermore, upon internalization of DXR-loaded NGO with a disulfide-linked PEG shell into HeLa cells, DXR is effectively released in the presence of an elevated GSH reducing environment, as observed in confocal microscopy and flow cytometric experiments. Importantly, inhibition of cell proliferation is directly correlated with increased intracellular GSH concentrations due to rapid DXR release.

Effective gene delivery using stimulus-responsive catiomer designed with redox-sensitive disulfide and acid-labile imine linkers.

A dual stimulus-responsive mPEG-SS-PLL(15)-glutaraldehyde star (mPEG-SS-PLL(15)-star) catiomer is developed and biologically evaluated. The catiomer system combines redox-sensitive removal of an external PEG shell with acid-induced escape from the endosomal compartment. The design rationale for PEG shell removal is to augment intracellular uptake of mPEG-SS-PLL(15)-star/DNA complexes in the presence of tumor-relevant glutathione (GSH) concentration, while the acid-induced dissociation is to accelerate the release of genetic payload following successful internalization into targeted cells. Size alterations of complexes in the presence of 10 mM GSH suggest stimulus-induced shedding of external PEG layers under redox conditions that intracellularly present in the tumor microenvironment. Dynamic laser light scattering experiments under endosomal pH conditions show rapid destabilization of mPEG-SS-PLL(15)-star/DNA complexes that is followed by facilitating efficient release of encapsulated DNA, as demonstrated by agarose gel electrophoresis. Biological efficacy assessment using pEGFP-C1 plasmid DNA encoding green fluorescence protein and pGL-3 plasmid DNA encoding luciferase as reporter genes indicate comparable transfection efficiency of 293T cells of the catiomer with a conventional polyethyleneimine (bPEI-25k)-based gene delivery system. These experimental results show that mPEG-SS-PLL(15)-star represents a promising design for future nonviral gene delivery applications with high DNA binding ability, low cytotoxicity, and high transfection efficiency.

Potential of Polyethyleneimine as an Adjuvant To Prepare Long-Term and Potent Antifungal Nanovaccine.

Background: Candida albicans infections are particularly prevalent in immunocompromised patients. Even with appropriate treatment with current antifungal drugs, the mortality rate of invasive candidiasis remains high. Many positive results have been achieved in the current vaccine development. There are also issues such as the vaccine's protective effect is not persistent. Considering the functionality and cost of the vaccine, it is important to develop safe and efficient new vaccines with long-term effects. In this paper, an antifungal nanovaccine with Polyethyleneimine (PEI) as adjuvant was constructed, which could elicit more effective and long-term immunity via stimulating B cells to differentiate into long-lived plasma cells. Materials and Methods: Hsp90-CTD is an important target for protective antibodies during disseminated candidiasis. Hsp90-CTD was used as the antigen, then introduced SDS to "charge" the protein and added PEI to form the nanovaccine. Dynamic light scattering and transmission electron microscope were conducted to identify the size distribution, zeta potential, and morphology of nanovaccine. The antibody titers in mice immunized with the nanovaccine were measured by ELISA. The activation and maturation of long-lived plasma cells in bone marrow by nanovaccine were also investigated via flow cytometry. Finally, the kidney of mice infected with Candida albicans was stained with H&E and PAS to evaluate the protective effect of antibody in serum produced by immunized mice. Results: Nanoparticles (NP) formed by Hsp90-CTD and PEI are small, uniform, and stable. NP had an average size of 116.2 nm with a PDI of 0.13. After immunizing mice with the nanovaccine, it was found that the nano-group produced antibodies faster and for a longer time. After 12 months of immunization, mice still had high and low levels of antibodies in their bodies. Results showed that the nanovaccine could promote the differentiation of B cells into long-lived plasma cells and maintain the long-term existence of antibodies in vivo. After immunization, the antibodies in mice could protect the mice infected by C. albicans. Conclusion: As an adjuvant, PEI can promote the differentiation of B cells into long-lived plasma cells to maintain long-term antibodies in vivo. This strategy can be adapted for the future design of vaccines.

Highly efficient drug delivery nanosystem via L-phenylalanine triggering based on supramolecular polymer micelles.

An intelligent drug delivery nanosystem has been developed based on biodegradable supramolecular polymer micelles (SMPMs). The drug release can be triggered from SMPMs responsively by a bioactive agent, L-phenylalanine in a controlled fashion. The SMPMs are constructed from ethylcellulose-graft-poly(ε-caprolactone) (EC-g-PCL) and α-cyclodextrin (α-CD) derivate via host-guest and hydrophobic interactions. It has been found that these SMPMs have disassembled rapidly in response to an additional L-phenylalanine, due to great affinity discrepancy to α-CD between L-phenylalanine and PCL. Experiments have been carried out on trigger-controlled in vitro drug release of the SMPMs loaded with a model porphyrin based photosensitizer THPP. The result shows that the SMPMs released over 85% THPP in 6 h, which is two orders magnitudes faster than that of control. Also investigated is the photodynamic therapy (PDT) of THPP-loaded SMPMs with and without L-phenylalanine on MCF-7 carcinoma cell line. An effective trigger-concentration dependent lethal effect has been found showing promise in clinical photodynamic therapy.

Engineered Biomimetic Nanoplatform Protects the Myocardium Against Ischemia/Reperfusion Injury by Inhibiting Pyroptosis.

Protection of cardiomyocytes against oxidative stress is vital to alleviate myocardial ischemia/reperfusion injury (MI/RI). However, antioxidative treatment is hampered by the lack of safe and effective therapeutics. Polydopamine (PDA), as a biodegradable class of nanomaterial with excellent antioxidant properties, has shown great potential in treating MI/RI. To achieve site-specific antioxidative efficacy, we established a PDA-based biomimetic nanoplatform (PDA@M), which consisted of a polydopamine core and a macrophage membrane shell to form a shell-core structure. By inheriting the inherent migration capability of macrophages, PDA@M was able to target the infarcted myocardium and exert an antioxidative effect to protect the myocardium. The results demonstrated that the accumulation of the membrane-wrapped nanoparticles (NPs) in the infarcted myocardium was greatly increased as compared with PDA alone, which effectively relieved the MI/RI-induced oxidative stress. PDA@M largely decreased the infarct size and improved the cardiac function post-MI/RI. Our study revealed that PDA@M could inhibit cell pyroptosis by suppressing the NLRP3/caspase-1 pathway, which is known to play a significant role in the antioxidant signaling pathway. In summary, PDA@M can target the infarcted myocardium and exert antioxidative and antipyroptosis functions to protect the myocardium against MI/RI-induced oxidative stress, suggesting that it may prove to be a potential therapeutic agent for MI/RI.

Ca2+-Mediated Surface Polydopamine Engineering to Program Dendritic Cell Maturation.

Engineering of cell surfaces holds promise in manipulating cellular activities in a physicochemical route as a complement to the biological approach. Mediated by Ca2+, a quick and convenient yet cytocompatible method is used to achieve surface engineering, by which polydopamine nanostructures can be in situ grown onto dendritic cell (DC) surfaces within 10 min. Ca2+, as the physical bridge between the negative cell surface and polydopamine, avoids the direct chemical polymerization of polydopamine onto the cell surface, critically important to maintain the cell viability. As a proof of concept in potential applications, this cell surface engineering shows a good control toward DC maturation. Upon surface polydopamine engineering, bone-marrow-derived DC exhibits a unique bidirectional control of maturation. The polydopamine structure enables effective suppression of DC activation by acting as an efficient scavenger of reactive oxygen species, a key signal during maturation. Conversely, an 808 nm laser irradiation can remotely relieve the suppressed state and effectively activate DC maturation by the photoheat effect of polydopamine (39 °C). The work provides an easily implemented, straightforward approach to achieve cell surface engineering, through which the DC maturation can be controlled.

Controlling the growth behaviour of multilayered films via layer-by-layer assembly with multiple interactions.

Layer-by-layer (LbL) assembly of chitosan-graft-phenyl (CHI-g-Ph) and poly(aspartic-graft-octadecyl) chain (PASP-g-OD) with multiple interactions, i.e. electrostatic interaction and hydrophobic interaction, was studied and the growth rate of multilayered films was found to be strongly dependent on the concentration of NaCl in the aqueous solution. LbL assembly of phenyl chitosan-graft-cyclodextrin (Ph-CHI-g-CD)/PASP-g-OD and chitosan-graft-cyclodextrin (CHI-g-CD)/PASP-g-OD with electrostatic interaction and host-guest interaction was further carried out. The growth of these multilayered films was also affected by the concentration of NaCl in the aqueous solution. For comparison, the LbL assembly of the chitosan (CHI)/polyaspartic acid (PASP) and CHI/PASP-g-OD multilayered films fabricated with electrostatic interaction only were also studied to identify the effect of the multiple interactions on the LbL assembly. (1)H MNR, transmission electron microscopy (TEM), fluorescence spectroscopy and dynamic light scattering (DLS) were used to characterize the polyelectrolytes. LbL assembly processes were monitored by UV-Vis spectroscopy, and the morphology of multilayered films was observed by atomic force microscopy (AFM). This research may provide a new strategy to control the growth behaviour of multilayered films via LbL assembly with multiple interactions.

Microfiber-Reinforced Composite Hydrogels Loaded with Rat Adipose-Derived Stem Cells and BMP-2 for the Treatment of Medication-Related Osteonecrosis of the Jaw in a Rat Model.

Severe adverse reactions of bisphosphonates and anti-resorptive or anti-angiogenic medications, termed medication-related osteonecrosis of the jaw (MRONJ), have been reported. MRONJ are difficult to completely cure and could cause great pain to patients. Recent studies have shown that mesenchymal stem cell (MSC) therapies are effective for treating MRONJ, but the method of intravenous injection is unstable and increases the risk of producing tumors. In the present study, low-acyl gellan gum (LAGG) hydrogels were modified with hemicellulose polysaccharide microfibers (PMs) to improve the performance of supporting three-dimensional (3D) cell growth. LAGG-PM composite hydrogels were found to be nontoxic to rat adipose-derived stem cells (rADSCs) in vitro. The hydrogels also promoted the secretion of angiogenic factors, induced osteoclastogenesis by conditioned medium, and supported osteogenic marker expression after the addition of human bone morphogenetic protein-2 (BMP-2). Due to its injectability, the LAGG-PM composite hydrogel incorporated with rADSCs and BMP-2 could be applied into the MRONJ lesion site, which promoted mucosal recovery, bone tissue reconstruction, and osteoclastogenesis. This study confirms the potential applications of LAGG-PM composite hydrogels as 3D cell culture platforms and delivery vehicles for the treatment of MRONJ in a rat model.

The synthesis of biodegradable graft copolymer cellulose-graft-poly(L-lactide) and the study of its controlled drug release.

According to the concept of green chemistry and sustainable development, a new biodegradable copolymer comprised of hydrophobic poly(l-lactide) (PLLA) segments and hydrophilic cellulose segment (cellulose-g-PLLA) was designed and synthesized. The structure of the copolymer was characterized by (1)H NMR, FT-IR, (13)C NMR, DSC and WAXD. The cytotoxicity study shows that cellulose-g-PLLA exhibits good biocompatibility. The copolymer can self-assemble into micelles in water with the hydrophobic PLLA segments at the cores of micelles and the hydrophilic cellulose segments as the outer shells. Transmission electron microscopy (TEM) shows that the micelles exhibit nanospheric morphology within a size range of 30-80nm. The drug loaded micelles formed by the copolymer in aqueous media show sustained drug release which indicates their potential applicability in drug carrier.

Polyethylene glycol-poly(ε-benzyloxycarbonyl-l-lysine)-conjugated VEGF siRNA for antiangiogenic gene therapy in hepatocellular carcinoma.

A polyethylene glycol-poly(ε-benzyloxycarbonyl-l-lysine) (PEG-SS-PLL) block copolymer based on a disulfide-linked, novel biodegradable catiomer bearing a PEG-sheddable shell was developed to avoid "PEG dilemma" in nanoparticle intracellular tracking of PEG-PLL where PEG was nondegradable. However, PEG-SS-PLL catiomers have not been used to deliver small interfering VEGF RNA (siVEGF) in antiangiogenesis gene therapy. In this study, we aimed to investigate whether this novel biodegradable catiomer can deliver siVEGF into cancer cells and at the same time have an antitumor effect in a xenograft mouse model. It was found that PEG-SS-PLL efficiently delivered siVEGF with negligible cytotoxicity, and significantly decreased the expression of VEGF at both the messenger-RNA and protein levels both in vitro and in vivo, and thus tumor growth was inhibited. Our findings demonstrated that PEG-SS-PLL/siVEGF could potentially be applied to antiangiogenesis gene therapy for hepatocellular carcinoma.

Thermosensitive Y-shaped micelles of poly(oleic acid-Y-N-isopropylacrylamide) for drug delivery.

A novel thermosensitive amphiphilic copolymer comprised of two hydrophobic poly(oleic acid) (POA) segments and one hydrophilic poly(N-isopropylacrylamide) (PNIPAAm) segment was designed and synthesized. The structure of the copolymer was confirmed as Y-shaped by FTIR, 1H NMR, and SEC-MALLS analysis. A cytotoxicity study shows that the P(OA-Y-NIPAAm) copolymer exhibits good biocompatibility. The copolymer may self-assemble into micelles in water, with the hydrophobic POA segments at the cores of micelles and the hydrophilic PNIPAAm segments as the outer shells. The resulting micelles demonstrate temperature sensitivity with a lower critical solution temperature (LCST) of 31.5 degrees C and a critical micelle concentration (CMC) of 12.6 mg L(-1). Transmission electron microscopy (TEM) shows that the micelles exhibit a nanospheric morphology within a narrow size range of approximately 10-30 nm. A study of controlled release reveals that the self-assembled micelles have great potential as drug carriers.

Disulfide-Bridged Cleavable PEGylation of Poly-L-Lysine for SiRNA Delivery.

Engineered PEG-cleavable catiomers based on poly-L-lysine have been developed as nonviral gene vectors, which have been found to be one of important methods to balance "PEG dilemma." In this protocol, we aim at the standardization of the method and procedure of PEG-cleavable catiomers. Major steps including ring-opening polymerization (ROP) of ε-benzyloxycarbonyl-L-lysine N-carboxyanhydride (zLL-NCA) monomers to yield PEG-cleavable polylysine, examination on bio-stability and bio-efficacy of its gene complexes are described.

Disulfide-bridged cleavable PEGylation in polymeric nanomedicine for controlled therapeutic delivery.

PEGylation in polymeric nanomedicine has gained substantial predominance in biomedical applications due to its resistance to protein absorption, which is critically important for a therapeutic delivery system in blood circulation. The shielding layer of PEGylation, however, creates significant steric hindrance that negatively impacts cellular uptake and intracellular distribution at the target site. This unexpected effect compromises the biological efficacy of the encapsulated payload. To address this issue, one of the key strategies is to tether the disulfide bond to PEG for constructing a disulfide-bridged cleavable PEGylation. The reversible disulfide bond can be cleaved to enable selective PEG detachment. This article provides an overview on the strategy, method and progress of PEGylation nanosystem with the cleavable disulfide bond.

Suppression of VEGF by reversible-PEGylated histidylated polylysine in cancer therapy.

A reversible-PEGylated polylysine is designed and developed for efficient delivery of siRNA. In this unique structure, the ε-amino groups of disulfide linked poly(ethylene glycol) (PEG) and polylysine (mPEG-SS-PLL) are partially replaced by histidine groups, in order to develop the histidylated reversible-PEGylated polylysine (mPEG-SS-PLH), for enhanced endosome escape ability. The transfection efficacy of mPEG-SS-PLH is found to closely correlate with histidine substitution. Its maximum transfection efficiencies are determined, respectively, to be 75%, 42%, and 24%, against 293T, MCF-7, and PC-3 cells. These data indicate that the transfection efficiencies can equal or even outweigh PEI-25k in the corresponding cells (80%, 38.5%, and 20%). The in vivo circulation and biodistribution of the polyplexes are monitored by fluorescent imaging. The in vivo gene transfection is carried out by intravenous injection of pEGFP to BALB/c mice using the xenograft models. The in vivo experimental results show effective inhibition of tumor growth by mPEG-SS-PLH/siRNA-VEGF, indicating its high potential for clinical applications.

Cleavable PEGylation and hydrophobic histidylation of polylysine for siRNA delivery and tumor gene therapy.

Polylysine with cleavable PEGylation and hydrophobic histidylation (mPEG-SS-Lysn-r-Hism) was designed and developed for efficient siRNA delivery and tumor therapy. mPEG-SS-Lysn-r-Hism was used to carry and deliver small interfering RNA (siRNA) for silencing endogenous vascular endothelial growth factor (VEGF) expression and inhibiting tumor growth in HepG2 tumor-bearing mice. In this gene vector, histidine(Bzl) was selected for hydrophobic histidylation for the proton sponge ability of the imidazole ring and hydrophobic benzyl group. Cleavable PEGylation was introduced for in vivo circulation as well as selective PEG detachment in response to intracellular reduction condition in order to release the genetic payload. PEG detachment induced gene release was supported by agarose gel electrophoresis retardation assay, undertaken in the intracellular relevant reduction condition. In vitro transfection evaluation of histidylated copolymers, using pEGFP as genetic model, indicated significantly higher GFP expression than unmodified counterparts, comparable to the gold standard PEI. The efficacy of hydrophobic histidylation was found to be pronounced in mesenchymal stem cells (MSCs). In vivo application of the VEGF-siRNA package by tailored mPEG-SS-Lysn-r-Hism showed distinct tumor suppression in terms of macroscopic tumor volume and molecular analysis.

Pluronic F127 nanomicelles engineered with nuclear localized functionality for targeted drug delivery.

PKKKRKV (Pro-Lys-Lys-Lys-Arg-Lys-Val, PV7), a seven amino acid peptide, has emerged as one of the primary nuclear localization signals that can be targeted into cell nucleus via the nuclear import machinery. Taking advantage of chemical diversity and biological activities of this short peptide sequence, in this study, Pluronic F127 nanomicelles engineered with nuclear localized functionality were successfully developed for intracellular drug delivery. These nanomicelles with the size ~100 nm were self-assembled from F127 polymer that was flanked with two PV7 sequences at its both terminal ends. Hydrophobic anticancer drug doxorubicin (DOX) with inherent fluorescence was chosen as the model drug, which was found to be efficiently encapsulated into nanomicelles with the encapsulation efficiency at 72.68%. In comparison with the non-functionalized namomicelles, the microscopic observation reveals that PV7 functionalized nanomicelles display a higher cellular uptake, especially into the nucleus of HepG2 cells, due to the nuclear localization signal effects. Both cytotoxicity and apoptosis studies show that the DOX-loaded nanomicelles were more potent than drug nanomicelles without nuclear targeting functionality. It was thus concluded that PV7 functionalized nanomicelles could be a potentially alternative vehicle for nuclear targeting drug delivery.