Injectable and Self-Curing Single-Component Hydrogel for Stem Cell Encapsulation and In Vivo Bone Regeneration.

An ideal hydrogel for stem cell therapy would be injectable and efficiently promote stem cell proliferation and differentiation in body. Herein, an injectable, single-component hydrogel with hyaluronic acid (HA) modified with phenylboronic acid (PBA) and spermidine (SM) is introduced. The resulting HAps (HA-PBA-SM) hydrogel is based on the reversible crosslinking between the diol and the ionized PBA, which is stabilized by the SM. It has a shear-thinning property, enabling its injection through a syringe to form a stable hydrogel inside the body. In addition, HAps hydrogel undergoes a post-injection "self-curing," which stiffens the hydrogel over time. This property allows the HAps hydrogel to meet the physical requirements for stem cell therapy in rigid tissues, such as bone, while maintaining injectability. The hydrogel enabled favorable proliferation of human mesenchymal stem cells (hMSCs) and promoted their differentiation and mineralization. After the injection of hMSCs-containing HAps into a rat femoral defect model, efficient osteogenic differentiation of hMSCs and bone regeneration is observed. The study demonstrates that simple cationic modification of PBA-based hydrogel enabled efficient gelation with shear-thinning and self-curing properties, and it would be highly useful for stem cell therapy and in vivo bone regeneration.

Inhibition of protein-protein interactions using biodegradable depsipeptide nanoassemblies.

Although peptides notoriously have poor intrinsic pharmacokinetic properties, it is well-known that nanostructures with excellent pharmacokinetic properties can be designed. Noticing that peptide inhibitors are generally nonpolar, here, we consolidate the peptide inhibitor targeting intracellular protein-protein interactions (PPIs) as an integral part of biodegradable self-assembled depsipeptide nanostructures (SdPNs). Because the peptide inhibitor has the dual role of PPI inhibition and self-assembly in this design, problems associated with the poor pharmacokinetics of peptides and encapsulation/entrapment processes can be overcome. Optimized SdPNs displayed better tumor targeting and PPI inhibition properties than the comparable small molecule inhibitor in vivo. Kinetics of PPI inhibition for SdPNs were gradual and controllable in contrast to the rapid inhibition kinetics of the small molecule. Because SdPN is modular, any appropriate peptide inhibitor can be incorporated into the platform without concern for the poor pharmacokinetic properties of the peptide.

Efficient drug supply in stem cell cytosol via pore-forming saponin nanoparticles promotes in vivo osteogenesis and bone regeneration.

Directional differentiation of stem cells is a key step in stem cell therapy. In this study, we developed saponin-based nanoparticles (Ad-SNPs) containing dexamethasone (Dex) and alpha-lipoic acid (ALA) to promote osteogenic differentiation of human mesenchymal stem cells (hMSCs) and bone regeneration. The Ad-SNPs can achieve rapid cellular uptake through a pore-forming effect without cytotoxic cationic charges. They also provide extended retention in cell cytosol due to their uptake route. These properties are advantageous in efficiently supplying drugs to the hMSCs. The combination of Dex and ALA facilitated mitochondrial fusion and prevented reactive oxygen species-induced DNA damage. It also helped to preserve mitochondrial dynamics, and the efficient supply of it provided by the Ad-SNPs induced differentiation of hMSCs into osteoblasts. The Ad-SNPs showed outstanding performance in osteoblast differentiation, maturation, and mineralization in 3D culture compared with NPs without saponin and with free drugs. When Ad-SNP-treated hMSCs were tested in a rat femoral bone defect model, they showed the fastest regeneration of bones and complete repair in the shortest period among all groups. To the best of our knowledge, this study is the first application of pore-forming saponin-based NPs with rapid cellular uptake and extended retention to stem cell therapy, and we demonstrated their promising potential in bone regeneration and efficient delivery of Dex and ALA.

ROS-responsive PEGylated ferrocene polymer nanoparticles with improved stability for tumor-selective chemotherapy and imaging.

Ferrocene-based nanoparticles have garnered interest as reactive oxygen species (ROS)-responsive nanocarriers of anticancer drugs and imaging agents. However, their biomedical applications remain limited due to their poor physiological stability. PEGylation of nanocarriers improves their stability and biocompatibility. In this study, we aimed to develop novel PEG-ferrocene nanoparticles (PFNPs) with enhanced stability and ROS responsiveness for the delivery of paclitaxel (PTX) and imaging agents. PEGylation improved the stability of ferrocene nanoparticles, inhibiting their ROS-responsive destruction. Several PEG-ferrocene polymers containing different molar ratios of methacrylic acid and poly (ethylene glycol) methyl ether methacrylate was designed for optimization. ROS-responsive polymers with optimal monomer ratios were self-assembled into PFNPs with enhanced stability. The PFNPs distended, effectively releasing encapsulated PTX and imaging agents within 8 h in the presence of ROS. Furthermore, they remained stable, with no changes in their hydrodynamic diameters or polydispersity indexes after storage in an aqueous solution and biological buffer. The accumulation of PFNPs in a tumor model in vivo was 15-fold higher than a free dye. PTX-loaded PFNPs showed a substantial tumor-suppression effect, reducing tumor size to approximately 18% of that in the corresponding control group. These findings suggest a promising application of ROS-responsive PFNPs in tumor treatment as biocompatible nanocarriers of anticancer drugs and imaging agents.

Organic and inorganic nanomedicine for combination cancer therapies.

In many cases, a single mode of cancer therapy shows limited efficacy in treating complex and heterogeneous tumors. To improve cancer treatment, combining chemo-, photodynamic-, photothermal-, radio-, and immunotherapy is clinically recognized. When different therapeutic treatments are combined, they often show synergetic effects that further improve therapeutic outcomes. In this review, we introduce nanoparticle (NP)-based combination cancer therapies that use organic and inorganic NPs. Liposomes, polymers, and exosomes can be prepared with amphiphilic properties, high physical stability, and low immune response to treat cancers in a multimodal way. Inorganic NPs, including upconversion, plasmonic, and mesoporous silica NPs, have emerged as a new technology for photodynamic-, photothermal-, and immunotherapy. These NPs can simultaneously carry multiple drug molecules and deliver them efficiently to tumor tissue, as demonstrated in many studies. In addition to reviewing recent advances in organic and inorganic NPs used in combination therapy for cancers, we also discuss their rational design and the outlook for future nanomedicine development.

Levan nanoparticles with intrinsic CD44-targeting ability for tumor-targeted drug delivery.

Existing anticancer therapeutics exhibit short half-lives, non-specificity, and severe side effects. To address this, active-targeting nanoparticles have been developed; however, the complex fabrication procedures, scale-up, and low reproducibility delay FDA approval, particularly for functionalized nanoparticles. We developed levan nanoparticles via simple one-pot nanoprecipitation for specific anticancer drug delivery. Levan is a plant polysaccharide which has a binding affinity to CD44 receptors and amphiphilicity. The nanoparticles are self-assembled and enable active-targeting without chemical modifications. The paclitaxel-loaded levan nanoparticles (PTX@LevNP) demonstrated a sustained PTX release and long-term stability. The LevNP can bind CD44 receptors on cancer cells, and PTX@LevNP showed enhanced anticancer activity in CD44-positive cells (SCC7 cells). In SCC7 tumor-bearing mice, the accumulation of LevNP in tumor tissue was 3.7 times higher than that of the free-dye, resulting in improved anticancer efficacy of PTX@LevNP. This new strategy using levan can produce nanoparticles for effective cancer treatment without complex fabrication procedures.

Recent advances in selective and targeted drug/gene delivery systems using cell-penetrating peptides.

Biological cell membranes are a natural barrier for living cells. In the last few decades, the cell membrane has been the main hurdle in the efficient delivery of bioactive and therapeutic agents. To increase the drug efficacy of these agents, additional mediators have been considered. Cell-penetrating peptides (CPPs), a series of oligopeptides composed of mostly hydrophobic and/or positively charged side chains, can increase the interaction with the cell membrane. CPP-based delivery platforms have shown great potential for the efficient and direct cytosol delivery of various cargos, including genes, proteins, and small molecule drugs. Bypassing endocytosis allows the CPP-based delivery systems greater defense against the degradation of protein-based drugs than other drug delivery systems. However, the delivery of CPPs exhibits intrinsically non-specific targeting, which limits their medical applications. To endow CPPs with specific targeting ability, the conjugation of pH-sensitive, enzyme-specific cleavable, and multiple targeting ligands has been reported. Optimization of the length and sequence of CPPs is still needed for various drugs of different sizes and surface charges. Toxicity issues in CPP-based delivery systems should be addressed carefully before clinical use.

Perfluorooctylbromide-loaded fucoidan-chlorin e6 nanoparticles for tumor-targeted photodynamic therapy.

Efficient delivery of a photosensitizer (PS) and oxygen to tumor tissue is critical for successful photodynamic therapy (PDT). For this purpose, we developed a fucoidan (Fu)-chlorin e6 (Ce6) nanoparticle (NP) containing perfluorooctylbromide (PFOB). Fu, a biopolymer derived from seaweed, made up the hydrophilic shell of the NP and provided specific targeting to tumor cells by P-selectin binding. Conjugation with the hydrophobic Ce6 enabled self-assembly and Ce6-generated cytotoxic reactive oxygen species to kill tumor cells upon laser irradiation. PF supplied oxygen to the hypoxic tumor tissue and increased the efficacy of the PDT. The developed Fu-Ce6-PF-NPs bound specifically to SCC7 tumor cells and killed them via a photodynamic effect on laser irradiation. High accumulation of the NPs in tumor tissue and improved tumor suppression by PDT were observed in SCC7 tumor-bearing mice. The overall data demonstrated the potential of Fu-Ce6-PF-NP as a tumor-targeting drug carrier for effective PDT.

Recent advances in optical imaging through deep tissue: imaging probes and techniques.

Optical imaging has been essential for scientific observations to date, however its biomedical applications has been restricted due to its poor penetration through tissues. In living tissue, signal attenuation and limited imaging depth caused by the wave distortion occur because of scattering and absorption of light by various molecules including hemoglobin, pigments, and water. To overcome this, methodologies have been proposed in the various fields, which can be mainly categorized into two stategies: developing new imaging probes and optical techniques. For example, imaging probes with long wavelength like NIR-II region are advantageous in tissue penetration. Bioluminescence and chemiluminescence can generate light without excitation, minimizing background signals. Afterglow imaging also has high a signal-to-background ratio because excitation light is off during imaging. Methodologies of adaptive optics (AO) and studies of complex media have been established and have produced various techniques such as direct wavefront sensing to rapidly measure and correct the wave distortion and indirect wavefront sensing involving modal and zonal methods to correct complex aberrations. Matrix-based approaches have been used to correct the high-order optical modes by numerical post-processing without any hardware feedback. These newly developed imaging probes and optical techniques enable successful optical imaging through deep tissue. In this review, we discuss recent advances for multi-scale optical imaging within deep tissue, which can provide reseachers multi-disciplinary understanding and broad perspectives in diverse fields including biophotonics for the purpose of translational medicine and convergence science. Methodologies for multi-scale optical imaging within deep tissues are discussed in diverse fields including biophotonics for the purpose of translational medicine and convergence science. Recent imaging probes have tried deep tissue imaging by NIR-II imaging, bioluminescence, chemiluminescence, and afterglow imaging. Optical techniques including direct/indirect and coherence-gated wavefront sensing also can increase imaging depth.

Structural control of self-assembled peptide nanostructures to develop peptide vesicles for photodynamic therapy of cancer.

Vesicles such as liposomes, polymersomes, and exosomes have been widely used as drug delivery carriers; however, peptide vesicles (peptidesomes) despite their potential utility are far less well developed. Peptidesomes are distinctive because peptides play dual roles as a self-assembly building block and a bioactive functional unit. In order for peptidesomes to become successful nanodrugs, the issues related to differences in nanostructural properties between in vitro and in vivo conditions should be addressed. Here, we delineate a multivariate approach to feedback control the structures of peptide building blocks, nanoparticle size, drug loading process, nanoparticle aggregation, cytotoxicity, cell targeting capability, endosome disruption function, protease resistance, and in vivo performance, which eventually enabled the successful development of a highly efficacious peptidesome for in vivo cancer therapy. This study lays the groundwork for the successful in vivo translation of peptide nanodrugs.

Methotrexate-loaded nanoparticles ameliorate experimental model of autoimmune arthritis by regulating the balance of interleukin-17-producing T cells and regulatory T cells.

BACKGROUND: Rheumatoid arthritis (RA) is a progressive systemic autoimmune disease that is characterized by infiltration of inflammatory cells into the hyperplastic synovial tissue, resulting in subsequent destruction of adjacent articular cartilage and bone. Methotrexate (MTX), the first conventional disease-modifying antirheumatic drug (DMARD), could alleviate articular damage in RA and is implicated in humoral and cellular immune responses. However, MTX has several side effects, so efficient delivery of low-dose MTX is important. METHODS: To investigate the efficacy of MTX-loaded nanoparticles (MTX-NPs) against experimental model of RA, free MTX or MTX-NPs were administered as subcutaneous route to mice with collagen-induced arthritis (CIA) at 3 weeks after CII immunization. The levels of inflammatory factors in tissues were determined by immunohistochemistry, confocal microscopy, real-time PCR, and flow cytometry. RESULTS: MTX-NPs ameliorated arthritic severity and joint destruction in collagen-induced arthritis (CIA) mice compared to free MTX-treated CIA mice. The levels of inflammatory cytokines, including interleukin (IL)-1ß, tumor necrosis factor-α, and vascular endothelial growth factor, were reduced in MTX-NPs-treated mice. Number of CD4 + IL-17 + cells decreased whereas the number of CD4 + CD25 + Foxp3 + cells increased in spleens from MTX- NPs-treated CIA mice compared to MTX-treated CIA mice. The frequency of CD19 + CD25 + Foxp3 + regulatory B cells increased in ex vivo splenocytes from MTX-loaded NPs-treated CIA mice compared to MTX-treated CIA mice. CONCLUSION: The results suggest that MTX-loaded NPs have therapeutic potential for RA.

Overcoming the obstacles of current photodynamic therapy in tumors using nanoparticles.

Photodynamic therapy (PDT) has been applied in clinical treatment of tumors for a long time. However, insufficient supply of pivotal factors including photosensitizer (PS), light, and oxygen in tumor tissue dramatically reduces the therapeutic efficacy of PDT. Nanoparticles have received an influx of attention as drug carriers, and recent studies have demonstrated their promising potential to overcome the obstacles of PDT in tumor tissue. Physicochemical optimization for passive targeting, ligand modification for active targeting, and stimuli-responsive release achieved efficient delivery of PS to tumor tissue. Various trials using upconversion NPs, two-photon lasers, X-rays, and bioluminescence have provided clues for efficient methods of light delivery to deep tissue. Attempts have been made to overcome unfavorable tumor microenvironments via artificial oxygen generation, Fenton reaction, and combination with other chemical drugs. In this review, we introduce these creative approaches to addressing the hurdles facing PDT in tumors. In particular, the studies that have been validated in animal experiments are preferred in this review over proof-of-concept studies that were only performed in cells.

Copper-Free Click Chemistry: Applications in Drug Delivery, Cell Tracking, and Tissue Engineering.

Traditionally, organic chemical reactions require organic solvents, toxic catalysts, heat, or high pressure. However, copper-free click chemistry has been shown to have favorable reaction rates and orthogonality in water, buffer solutions, and physiological conditions without toxic catalysts. Strain-promoted azide-alkyne cycloaddition and inverse electron-demand Diels-Alder reactions are representative of copper-free click chemistry. Artificial chemical reactions via click chemistry can also be used outside of the laboratory in a controllable manner on live cell surfaces, in the cytosol, and in living bodies. Consequently, copper-free click chemistry has many features that are of interest in biomedical research, and various new materials and strategies for its use have been proposed. Herein, recent remarkable trials that have used copper-free click chemistry are described, focusing on their applications in molecular imaging and therapy. The research is categorized as nanoparticles for drug delivery, imaging agents for cell tracking, and hydrogels for tissue engineering, which are rapidly advancing fields based on click chemistry. The content is based primarily on the experience with click chemistry-based biomaterials over the last 10 years.

Rhamnolipid-coated W/O/W double emulsion nanoparticles for efficient delivery of doxorubicin/erlotinib and combination chemotherapy.

BACKGROUND: Combination therapy using more than one drug can result in a synergetic effect in clinical treatment of cancer. For this, it is important to develop an efficient drug delivery system that can contain multiple drugs and provide high accumulation in tumor tissue. In particular, simultaneous and stable loading of drugs with different chemical properties into a single nanoparticle carrier is a difficult problem. RESULTS: We developed rhamnolipid-coated double emulsion nanoparticles containing doxorubicin and erlotinib (RL-NP-DOX-ERL) for efficient drug delivery to tumor tissue and combination chemotherapy. The double emulsion method enabled simultaneous loading of hydrophilic DOX and hydrophobic ERL in the NPs, and biosurfactant RL provided stable surface coating. The resulting NPs showed fast cellular uptake and synergetic tumor cell killing in SCC7 cells. In real-time imaging, they showed high accumulation in SCC7 tumor tissue in mice after intravenous injection. Furthermore, enhanced tumor suppression was observed by RL-NP-DOX-ERL in the same mouse model compared to control groups using free drugs and NPs containing a single drug. CONCLUSIONS: The developed RL-NP-DOX-ERL provided efficient delivery of DOX and ERL to tumor tissue and successful tumor therapy with a synergetic effect. Importantly, this study demonstrated the promising potential of double-emulsion NPs and RL coating for combination therapy.

A dimeric α-helical cell penetrating peptide mounted with an HER2-selective affibody.

We have developed a cell penetrating peptide (CPP) system with high selectivity and penetrability at nanomolar concentrations with a combination of an HER2-selective affibody, ZHER2:342 (ZHER2), and a dimeric α-helical leucine- and lysine-rich peptide, LK-2. ZHER2 and LK-2 are linearly fused together and expressed in a prokaryotic system to create the LK-2-ZHER2 protein, which can successfully distinguish and penetrate HER2-overexpressing cancer cells at nanomolar concentrations. LK-2-ZHER2 has the ability to intracellularly deliver doxorubicin as a conjugate form to enhance its anti-cancer effect on HER2-overexpressing breast cancer cells with a great selectivity. The selective penetrability was confirmed in vitro, in 3D spheroids, and in in vivo models. LK-2-ZHER2 has the capability to overcome the weak points of current CPPs, such as poor penetrability at low concentrations and a lack of selectivity, by combining powerful CPP and affibody sequences.

Optimized Combination of Photodynamic Therapy and Chemotherapy Using Gelatin Nanoparticles Containing Tirapazamine and Pheophorbide a.

In combination therapy, synergetic effects of drugs and their efficient delivery are essential. Herein, we screened 12 anticancer drugs for combination with photodynamic therapy (PDT) using pheophorbide a (Pba). On the basis of combination index (CI) values in cell viability tests, we selected tirapazamine (TPZ) and developed self-assembled gelatin nanoparticles (NPs) containing both Pba and TPZ. The resulting TPZ-Pba-NPs showed a synergetic effect to kill tumor cells because TPZ was activated under the hypoxic conditions that originated from the PDT with Pba and laser irradiation. After they were injected into tumor-bearing mice via the tail vein, TPZ-Pba-NPs showed 3.17-fold higher blood concentration and 4.12-fold higher accumulation in tumor tissue 3 and 24 h postinjection, respectively. Upon laser irradiation to tumor tissue, TPZ-Pba-NPs successfully suppressed tumor growth by efficient drug delivery and synergetic effects in vivo. These overall results suggest that in vitro screening of drugs based on CI values, mechanism studies in hypoxia, and real-time in vivo imaging are promising strategies in developing NPs for optimized combination therapy.

Intratympanic administration of alpha-lipoic acid-loaded pluronic F-127 nanoparticles ameliorates acute hearing loss.

We used antioxidant-containing nanoparticles (NPs) to treat acute hearing loss. Alpha-lipoic acid (ALA) served as the antioxidant; we employed Pluronic F127 to fabricate NPs. In vitro, ALA-NPs protected cells of the organ of Corti in HEI-OC1 mice, triggering nuclear translocation of NRF2 and increases in the levels of antioxidant proteins, including Nrf2, HO-1, SOD-1, and SOD-2. In vivo, the hearing of mice that received ALA-NP injections into the middle ear cavity was better preserved after induction of ototoxicity than in control animals. The cochlear Nrf2 level increased in test mice, indicating that the ALA-NPs protected hearing via the antioxidant mechanism observed in vitro. ALA-NPs effectively protected against acute hearing loss by activating the Nrf2/HO-1 pathway.

In vivo vocal fold augmentation using an injectable polyethylene glycol hydrogel based on click chemistry.

It is important to focus on urgent needs in clinics and develop optimal materials. For successful augmentation of vocal folds, the ideal filler should be injectable through a syringe, and should stably maintain its volume for a long time without toxicity. To achieve these criteria, a click chemistry-based PEG (polyethylene glycol) hydrogel was developed and applied for vocal fold augmentation in vivo. The PEG hydrogel enables fast gelation in vivo after injection and provides long-term stability. Azide- and dibenzocyclooctyne (DBCO)-modified 4-arm PEG were cross-linked by chemical conjugation via click chemistry and yielded gelation within several minutes. After subcutaneous injection into mice and rats, the PEG hydrogel showed higher stability after 1 month compared to the traditionally used calcium hydroxyapatite-carboxymethyl cellulose (CaHA-CMC) filler. In rabbit models with vocal fold paralysis, the PEG hydrogel stably fixed the paralyzed vocal fold in 4 months and minimized the glottic gap. It was an improved therapeutic result compared to CaHA-CMC, demonstrating the potential of a click chemistry-based PEG hydrogel for vocal fold therapy.

Selection and identification of a novel bone-targeting peptide for biomedical imaging of bone.

The global burden of bone-related diseases is increasing in the aging society; thus, improved bone targeted imaging for their early identification and treatment are needed. In this study, we screened novel peptide ligands for hydroxyapatite, a major inorganic component of teeth and bones, and identified a peptide enabling in vivo bone targeting and real-time fluorescence bone detection. To isolate peptides highly specific for hydroxyapatite, we used negative and positive selection from a randomized 8-mer peptide phage library and identified hydroxyapatite-specific peptides (HA-pep2, HA-pep3, and HA-pep7). Among these three peptides, HA-pep3 showed the highest binding capacity and superior dissociation constant towards hydroxyapatite surfaces over time (~ 88.3% retained on hydroxyapatite after two weeks). Furthermore, HA-pep3 was highly specific for hydroxyapatite compared to other calcium salt-based materials. Using this superior specificity, HA-pep3 showed higher accumulation in skull, spine, and joints in comparison with scrambled control peptide during real-time whole-body imaging. Ex vivo analysis of the major organs and bone from mice demonstrated that the fluorescence intensity in bone was about 3.32 folds higher in the case of HA-pep3 than the one exhibited by the scrambled control peptide. Our study identified a novel approach for targeting ligands for bone specific imaging and can be useful for drug delivery applications.

A Dodecapeptide Selected by Phage Display as a Potential Theranostic Probe for Colon Cancers.

AIM: Colon cancer is one of the leading causes of cancer-related mortality. However, specific biomarkers for its diagnosis or treatment are not established well. METHODS: We developed a colon-cancer specific peptide probe using phage display libraries. We validated the specificity of this probe to colon cancer cells with immunohistochemical staining and FACS analysis using one normal cell and five colon cancer cell lines. RESULTS: This peptide probe maintained binding affinity even after serum incubation. For therapeutic applications, this peptide probe was conjugated to hematoporphyrin, a photosensitizer, which showed a significantly enhanced cellular uptake and high photodynamic effect to kill tumor cells. As another application, we made a nanoparticle modified from the peptide probe. It efficiently delivered SN-38, an anticancer drug, into tumor cells, and its tumor-targeting ability was observed in vivo after intravenous injection to the same xenograft model. CONCLUSION: The noble dodecapeptide probe can be a promising candidate for both colon tumor diagnosis and targeted drug delivery.