Chih-Chang Chu

Triple-negative breast cancer (TNBC) frequently evades immune recognition and elimination, resulting in an immunosuppressive microenvironment. The phagocytic activity of tumor-associated macrophages underscores the development of nanomaterials as a promising strategy to target these macrophages and modulate their polarization, thereby advancing immunotherapy against TNBC. This research developed functional polymers that are complexed with therapeutic molecules as a coating strategy for iron oxide nanoparticles. An arginine-based poly (ester urea urethane) polymer complexed with a macrophage-polarizing molecule (APU-R848) could provide a synergistic effect with iron oxide nanoparticles (IONPs) to stimulate the M1-polarization of macrophages at the tumor site, resulting in a versatile nano-platform for immune regulation of TNBC. In the 4T1 in vivo breast tumor model, the APU-R848-IONPs demonstrated an improved intratumoral biodistribution compared to IONPs without a polymer coating. APU-R848-IONPs significantly reversed the immune-suppressive tumor environment by reducing the M2/M1 macrophage phenotype ratio by 51%, associated with an elevated population of cytotoxic T cells and a significantly enhanced production of tumoricidal cytokines. The activated immune response induced by APU-R848-IONP resulted in a significant anti-tumor effect, demonstrating an efficacy that was more than 3.2-fold more efficient compared to the controls. These immune-regulatory pseudo-protein-coated iron oxide nanoparticles represent an effective nano-strategy for macrophages’ regulation and the activation of anti-tumor immunity, providing a new treatment modality for triple-negative breast cancer.

Pro-regenerative lipid mediator 1 (PreM1) is a specialized pro-resolving lipid mediator that promotes wound healing and regenerative functions of mesenchymal stem cells (MSCs), endothelial cells, and macrophages. The healing of third-degree (3°) burns and regenerative functions of MSCs are enhanced by ACgel1, an arginine-and-chitosan-based protein-mimic hybrid hydrogel. Adipose-tissue derived microvascular fragments (MVFs) are native vascularization units and a rich source of MSCs, endothelial cells, and perivascular cells for tissue regeneration. Here we describe an innovative PreM1-MVFs-ACgel1 construct that incorporated PreM1 and MVFs into ACgel1 via optimal design and fabrication. This construct delivered PreM1 to 3°-burn wounds at least up to 7 days-post-burn (dpb), and scaffolded and delivered MVFs. PreM1-MVFs-ACgel1 promoted the healing of 3°-burns in mice, including vascularization and collagen formation. The re-epithelization and closure of 3° burn wounds were promoted by ACgel1, MVFs, PreM1, MVFs-ACgel1, PreM1-ACgel1, or PreM1-MVFs-ACgel1 at certain time-point(s), while PreM1-MVFs-ACgel1 was most effective with 97% closure and 4.69% relative epithelial gap at 13 dpb compared to saline control. The PreM1-ACgel1 and MVFs-ACgel1 also promoted blood vessel regeneration of 3°-burns although PreM1-MVFs-ACgel1 is significantly more effective. These PreM1- and/or MVF-functionalized ACgel1 have nonexistent or minimal graft-donor requirements and are promising adjuvant therapeutic candidates for treating deep burns.

Pro-regenerative lipid mediator 1 (PreM1) is a specialized pro-resolving lipid mediator capable of promoting wound healing and regenerative functions of mesenchymal stem cells (MSCs), en-dothelial cells, and macrophages. The third-degree (3o) burn healing and regenerative functions of MSCs are also enhanced by ACgel1, an arginine and chitosan-based protein-mimic hybrid hy-drogel. Adipose-tissue derived microvascular fragments (MVFs) are native vascularization units and a rich source of MSCs, endothelial cells, and perivascular cells for tissue regeneration. Here we describe an innovative PreM1-MVFs-ACgel1 construct that incorporates PreM1 and MVFs into ACgel1 via optimal design and fabrication is capable of delivering PreM1 to 3o burn wounds at least up to 7 days post-burn (dpb), and of scaffolding and delivering MVFs, which promoted 3o burn the healing in mice, including vascularization and collagen formation. The re-epithelization and closure of 3o burn wounds were promoted by ACgel1, MVFs, PreM1, MVFs-ACgel1, PreM1-ACgel1, or PreM1-MVFs-ACgel1 at certain time-point(s) while PreM1-MVFs-ACgel1 was most effective. The PreM1-ACgel1 and MVFs-ACgel1 also promoted blood vessel regeneration of the 3o burns. These PreM1- and/or MVFs- functionalized ACgel1 have nonexistent or minimal graft-donor requirements and are promising adjuvant therapies for overcoming the drawbacks of current skin grafting or substituting for treating severe deep burns.

By exploring the structural design and tunability of polymeric nanocarriers, amino acid-based poly(ester urea urethane) is developed as a delivery vehicle for gambogic acid in the treatment of triple negative breast cancer.

Triple negative breast cancer (TNBC) has the worst overall survival among all breast cancer subtypes; 80% of TNBC harbors TP53 mutation. Gambogic acid (GA) is an herbal compound isolated from the dry brownish gamboge resin of Garcinia hanburyi. A new family of biodegradable polymer, the folate (FA)-conjugated arginine-based poly(ester urea urethane)s nanoparticles (FA-Arg-PEUU NP), was developed as nano-carrier for GA. Its anti-TNBC effects and the underlying mechanism of action were examined. The average diameters of FA-Arg-PEUU NP and GA-loaded FA-Arg-PEUU NP (NP-GA) in water are around 165 and 220nm, respectively. Rhodamine-tagged FA-Arg-PEUU NP shows that the conjugation of FA onto Arg-PEUU NPs facilitates the internalization of FA-Arg-PEUU-NP into TNBC. Compared to free-GA at the same GA concentrations, NP-GA exhibits higher cytotoxicity in both TP53-mutated and non-TP53 expressed TNBC cells by increasing intrinsic and extrinsic apoptosis. In HCC1806-bearing xenograft mouse model, the targeted delivery of GA by the FA-Arg-PEUU-NP nano-carriers to the tumor sites results in a more potent anti-TNBC effect and lower toxicity towards normal tissues and organs when compared to free GA. Furthermore, NP-GA also reduces the tumor-associated macrophage (TAM) M1/M2 ratio, suggesting that the use of Arg-based nanoparticles as carriers for GA not only makes the surface of the nanoparticles positively charged, but also confers on to the nanoparticles an ability to modulate TAM polarization. Our data clearly demonstrate that NP-GA exhibits potent anti-TNBC effects with reduced off-target toxicity, which represents novel alternative targeted therapeutics for TNBC treatment.

Reactive oxygen species (ROS) play an important role in cellular metabolism and many oxidative stress related diseases. Oxidative stress results from toxic effects of ROS and plays a critical role in the pathogenesis of a variety of diseases like cancers and many important biological processes. It is known that the unique feature of high intracellular ROS level in cancer cells can be considered as target and utilized as a useful cancer‐related stimulus to mediate intracellular drug delivery. Therefore, biomaterials responsive to excess level of ROS are of great importance in biomedical applications. In this study, a novel ROS‐responsive polymer based on L‐methionine poly(ester amide) (Met‐PEA‐PEG) was designed, synthesized, characterized and self‐assembled into nano‐micellar‐type nanoparticles (NP). The Met‐PEA‐PEG NP exhibited responsiveness to an oxidative environment. The size and morphology of the nanoparticle changed rapidly in the presence of H₂O₂. The Nile Red dye was loaded into the Met‐PEA‐PEG NP to demonstrate a H₂O₂ concentration induced time‐dependent release behavior. The Met‐PEA‐PEG NP was sensitive to high intracellular ROS level of PC3 prostate cancer cells. Furthermore, the Met‐PEA‐PEG NP was investigated as a carrier of a Chinese medicine‐based anticancer component, gambogic acid (GA). Compared to free GA, the GA‐loaded nanocomplex (GA‐NP) showed enhanced cytotoxicity toward PC3 and HeLa cells. The GA‐NP also induced a higher level of apoptosis and mitochondrial depolarization in PC3 cells than free GA. The Met‐PEA‐PEG NP improved the therapeutic effect of GA and may serve as a potential carrier for anticancer drug delivery.

Reduction-sensitive nanomedicine is a promising strategy to achieve controlled release of payloads in response to intracellular reductive milieu. However, endolysosomal sequestration of internalized carriers and insufficient redox potential in endolysosomes may delay the release of payloads and impact their therapeutic efficacy. Photochemical internalization (PCI), which takes advantage of light-induced endolysosomal rupture, is an effective technique for endosomal escape and cytosolic release of cargos. In this study, a biodegradable and reduction-sensitive nanocomplex was developed from arginine based poly(ester amide)s and hyaluronic acid (HA), and the PCI-photosensitizer AlPcS2a was conjugated to the surface of the nanocomplex (ArgPEA-ss-HA(AP)). This nanocomplex was used for the co-delivery of both PCI-photosensitizers and therapeutic agents to eliminate the biodistribution discrepancy resulting from the separated administration of free therapeutics. The PCI effect of the ArgPEA-ss-HA(AP) nanocomplex was validated in both monolayers and 3D spheroid models of MDA-MB-231 breast cancer cells. Synergism was detected between the PCI effect and doxorubicin-loaded nanocomplex in the inhibition of MDA-MB-231 cells. In addition, the ArgPEA-ss-HA(AP) nanocomplex also provided enhanced intratumoral penetration in 3D spheroids compared to free AlPcS2a. The in vivo results suggested that the conjugation of AlPCs2a in the nanocomplex enabled the consistent and preferential accumulation of both doxorubicin and AlPcS2a in tumor sites. A light-enhanced anti-tumor effect was observed for the doxorubicin-loaded nanocomplex at well-tolerable dosage. The ArgPEA-ss-HA(AP) nanocomplex, as a reduction-responsive delivery vehicle, can hold great potential to achieve spatio-temporally controllable anti-tumor effects.

Severe burns are some of the most challenging problems in clinics and still lack ideal modalities. Mesenchymal stem cells (MSCs) incorporated with biomaterial coverage of burn wounds may offer a viable solution. In this report, we seeded MSCs to a biodegradable hybrid hydrogel, namely ACgel, that was synthesized from unsaturated arginine-based poly(ester amide) (UArg-PEA) and chitosan derivative. MSC adhered to ACgels. ACgels maintained a high viability of MSCs in culture for 6 days. MSC seeded to ACgels presented well in third-degree burn wounds of mice at 8 days post-burn (dpb) after the necrotic full-thickness skin of burn wounds was debrided and filled and covered by MSC-carrying ACgels. MSC-seeded ACgels promoted the closure, re-epithelialization, granulation tissue formation, and vascularization of the burn wounds. ACgels alone can also promote vascularization but less effectively compared to MSC-seeded ACgels. The actions of MSC-seeded ACgels or ACgels alone involve the induction of reparative, anti-inflammatory interleukin-10 and M2-like macrophages, as well as the reduction of inflammatory cytokine TNFα and M1-like macrophages at the late inflammatory phase of burn-wound healing, which provided the mechanistic insights associated with inflammation and macrophages in burn wounds. For the studied regimens of these treatments, no toxicity was identified to MSCs or mice. Our results indicate that MSC-seeded ACgels have potential use as a novel adjuvant therapy for severe burns to complement commonly used skin grafting and, thus, minimize the downsides of grafting.