Airway epithelial-targeted nanoparticle reverses asthma in inhalation therapy.

Despite extensive research on corticosteroids for treating asthma, their short residence time in the lungs has limited their therapeutic effects in vivo. Nanoparticles have been widely investigated for inhaled drug delivery due to their potential benefits in prolonging drugs' residence time in the lungs. However, the retention of nanoparticles may be limited by mucus and ciliated epithelium clearance mechanisms in the airway. Herein, we anchored a neonatal-Fc-receptor-targeted peptide (FcBP) onto "mucus-penetrating" polyethylene glycol (PEG) nanoparticles (PEG-NP). Interestingly, the mucus-permeability of PEG-NP was not impaired by FcBP-functionalization. Moreover, FcBP modification enhanced cellular internalization and exocytosis via specific receptor-mediated processes, which subsequently ameliorated transepithelial transport and prolonged pulmonary retention. Importantly, after loading dexamethasone, FcBP-functionalization could effectively help nanoparticles cross the airway epithelial layer and be endocytosed by inflammatory cells, resulting in a marked decrease in inflammatory cytokines. Finally, FcBP modification significantly enhanced the therapeutic effect of dexamethasone-loaded nanoparticles in asthma mice. This study demonstrates that FcBP-functionalized PEG-NP can overcome multiple obstacles in the airway to prolong the pulmonary retention of drugs, providing a promising strategy for inhalation therapy.

Disease-specific protein corona formed in pathological intestine enhances the oral absorption of nanoparticles.

Protein corona (PC) has been identified to impede the transportation of intravenously injected nanoparticles (NPs) from blood circulation to their targeted sites. However, how intestinal PC (IPC) affects the delivery of orally administered NPs are still needed to be elucidated. Here, we found that IPC exerted "positive effect" or "negative effect" depending on different pathological conditions in the gastrointestinal tract. We prepared polystyrene nanoparticles (PS) adsorbed with different IPC derived from the intestinal tract of healthy, diabetic, and colitis rats (H-IPC@PS, D-IPC@PS, C-IPC@PS). Proteomics analysis revealed that, compared with healthy IPC, the two disease-specific IPC consisted of a higher proportion of proteins that were closely correlated with transepithelial transport across the intestine. Consequently, both D-IPC@PS and C-IPC@PS mainly exploited the recycling endosome and ER-Golgi mediated secretory routes for intracellular trafficking, which increased the transcytosis from the epithelium. Together, disease-specific IPC endowed NPs with higher intestinal absorption. D-IPC@PS posed "positive effect" on intestinal absorption into blood circulation for diabetic therapy. Conversely, C-IPC@PS had "negative effect" on colitis treatment because of unfavorable absorption in the intestine before arriving colon. These results imply that different or even opposite strategies to modulate the disease-specific IPC need to be adopted for oral nanomedicine in the treatment of variable diseases.

Cell or cell derivative-laden hydrogels for myocardial infarction therapy: from the perspective of cell types.

Myocardial infarction (MI) is a global cardiovascular disease with high mortality and morbidity. To treat acute MI, various therapeutic approaches have been developed, including cells, extracellular vesicles, and biomimetic nanoparticles. However, the clinical application of these therapies is limited due to low cell viability, inadequate targetability, and rapid elimination from cardiac sites. Injectable hydrogels, with their three-dimensional porous structure, can maintain the biomechanical stabilization of hearts and the transplantation activity of cells. However, they cannot regenerate cardiomyocytes or repair broken hearts. A better understanding of the collaborative relationship between hydrogel delivery systems and cell or cell-inspired therapy will facilitate advancing innovative therapeutic strategies against MI. Following that, from the perspective of cell types, MI progression and recent studies on using hydrogel to deliver cell or cell-derived preparations for MI treatment are discussed. Finally, current challenges and future prospects of cell or cell derivative-laden hydrogels for MI therapy are proposed.

Advancing immune checkpoint blockade in colorectal cancer therapy with nanotechnology.

Immune checkpoint blockade (ICB) has gained unparalleled success in the treatment of colorectal cancer (CRC). However, undesired side effects, unsatisfactory response rates, tumor metastasis, and drug resistance still hinder the further application of ICB therapy against CRC. Advancing ICB with nanotechnology can be game-changing. With the development of immuno-oncology and nanomaterials, various nanoplatforms have been fabricated to enhance the efficacy of ICB in CRC treatment. Herein, this review systematically summarizes these recent nano-strategies according to their mechanisms. Despite their diverse and complex designs, these nanoplatforms have four main mechanisms in enhancing ICB: 1) targeting immune checkpoint inhibitors (ICIs) to tumor foci, 2) increasing tumor immunogenicity, 3) remodeling tumor microenvironment, and 4) pre-sensitizing immune systems. Importantly, advantages of nanotechnology in CRC, such as innovating the mode-of-actions of ICB, modulating intestinal microbiome, and integrating the whole process of antigen presentation, are highlighted in this review. In general, this review describes the latest applications of nanotechnology for CRC immunotherapy, and may shed light on the future design of ICB platforms.

[Construction of Oral Insulin-Loaded Solid Lipid Nanoparticles and Their Intestinal Epithelial Cell Transcytosis Study].

OBJECTIVE: To construct solid lipid nanoparticle (SNPs) drug delivery system loaded with peptide and protein drugs by using mixedsolvents, to study the transcytosis mechanisms of SNPs across intestinal epithelial cells, and to improve the endocytosis and transcytosis efficiency of peptide and protein drugs. METHODS: The formulation of insulin-loaded water-in-oil-in-water solid lipid nanoparticles (INS-SNPs) was prepared by using a methanol-chloroform mixed solvent. The formulation was optimized with the single factor screening method. The optimized INS-SNPs were then characterized in terms of their morphology, stability and drug release properties. The cytotoxicity, cellular uptake and endocytosis mechanisms of INS-SNPs were then assessed on Caco-2 cells. The transcytosis efficiency of INS-SNPs was finally evaluated by using cellular monolayer in Transwell ® insert. RESULTS: The size, zeta potentials and drug loading efficiency of the optimized INS-SNPs were observed to be (145.4±0.5) nm, (-12.9±0.2) mV and (7.93±0.02)%, respectively. INS-SNPs were then shown to maintain desirable colloidal stability and sustained release of insulin in the simulated intestinal fluid. It was revealed that the cellular uptake of INS-SNPs reached its maximum after cellular incubation for 2 hours and was 1.53-fold higher than that of free insulin. Investigation of the endocytic mechanism revealed that INS-SNPs enter intestinal epithelial cells mainly through the clathrin-mediated and caveolae-mediated endocytosis pathways. Further investigation revealed that the amount of INS-SNPs permeating the cell monolayers was 1.54-fold higher than that of free insulin, which was comparable to the increase in their cellular uptake efficiency, indicating that INS-SNPs displayed enhanced absorption across the intestinal epithelium. CONCLUSION: The INS-SNPs prepared with mixed solvents in this study could significantly enhance the transcytosis efficiency of peptide and protein drugs, displaying great potentials in the application of oral drug delivery. This study may provide information and reference for the designing of efficient oral nano-drug delivery system in the future.

Complying with the physiological functions of Golgi apparatus for secretory exocytosis facilitated oral absorption of protein drugs.

Intestinal epithelial cells are the primary biological barriers for orally administrated nano-formulations and the delivered protein drugs. Thereinto, besides the cellular uptake, intracellular trafficking pathway and the related exocytosis are of great importance to the trans-epithelial transport of drug-loaded NPs. Herein, inspired by the physiological functions of Golgi apparatus for secreting proteins out of cells, Golgi localization-related amino acid l-cysteine (Cys) was modified on the surface of NPs to see whether and how this modification could guide the Golgi pathway-related transport and facilitate the exocytosis of drug-loaded NPs. Meanwhile, cell-penetrating peptide octa-arginine (R8) was co-modified to increase the cellular uptake. The proportion of R8 and Cys modification was explored to get the best effect of endocytosis and exocytosis of NPs. As a result, 25%R8 + 75%Cys NPs with most Cys modification showed efficient transcytosis with the highest transcytosis/endocytosis ratio (0.87). Interestingly, exocytosis mechanism studies indicated that they trafficked through the Golgi secretory pathway and bypassed lysosomes due to Cys modification. The detailed Golgi position mechanism studies further suggested that the thiol group from Cys was important for mediating Golgi transport. In particular, competitive inhibition studies demonstrated that Cys-modified NPs were more conducive to their exocytosis after being transported through the Golgi secretory pathway. We proved that cargos transported via Golgi apparatus tended to be trafficked out of the cells and avoid degradation, which contributed to the transcytosis of 25%R8 + 75%Cys NPs in vitro. Inspiringly, compared with unmodified NPs, 25%R8 + 75%Cys NPs also exhibited promoted intestinal penetration and oral absorption in vivo. Oral delivery of insulin-loaded 25%R8 + 75%Cys NPs showed stronger hypoglycemic effects in diabetic rats. In summary, this work provides a strategy for complying with the physiological functions of Golgi apparatus for secreting to facilitate the exocytosis of NPs, thus further improving the oral absorption of loaded protein drugs.

Tailored elasticity combined with biomimetic surface promotes nanoparticle transcytosis to overcome mucosal epithelial barrier.

Overcoming epithelial barriers to enhance drug absorption is a major challenge for nanoparticle (NP)-based mucosal delivery systems. With adequate physicochemical properties, the transepithelial delivery of NPs may be efficiently enhanced. However, little is known about the role of elasticity on the transport of NPs across the polarized epithelium, especially the processes and mechanisms of endocytosis, intracellular trafficking and exocytosis. In this study, we discovered that zwitterionic hydrogel NPs with varied elasticity displayed considerably different oral insulin absorption on diabetic rats. It was found that NP elasticity strongly shaped the transepithelial behaviors of NPs, and the increase of elasticity boosted the transcytosis by improving both endocytosis and exocytosis. Elasticity also showed a profound effect on the intracellular trafficking routes of NPs, which was closely related to distribution of NPs in exocytosis pathway and their intra-endosome sphere-to-ellipsoid shape transformation. Importantly, NPs with zwitterionic surface experienced more efficient basolateral exocytosis than apical exocytosis, while the elasticity-related exocytosis enhancement appeared to be non-selective. Therefore, tailored elasticity could promote mucosal transcytosis of NPs, which was able to be further improved with biomimetic zwitterionic surface. This study may provide important knowledge for the design of functional nanovehicles to efficiently overcome mucosal epithelial barriers in the future.

Nanoparticles with surface features of dendritic oligopeptides as potential oral drug delivery systems.

Surface features are key to the transcellular transport of nanoparticles (NPs) across intestinal epithelium cells. Endowing the NPs with specific surface features adapted to the physiological conditions of the gastrointestinal (GI) tract holds great potential for the oral delivery of peptide/protein drugs. Therefore, in this work, a glutamic acid conjugated amphiphilic dendrimer (Glu-APD) was synthesized to replace the widely used 1,2-distearoyl-sn-glycero-3-phosphatidyl-ethanolamine-polyethylene glycol (DSPE-PEG) in the preparation of poly(lactic-co-glycolic acid) (PLGA)-based NPs. Glu-APD could provide the formed NPs (Glu-APD NPs) with specific surface features of dendritic oligopeptides. With such surface features, Glu-APD NPs exhibited a 7.78-fold increase in cellular uptake and a 2.17-fold increase in the transepithelial transport amount compared with those of the DSPE-PEG2000 modified counterparts (P NPs). Instead of a dominant clathrin-mediated endocytosis as shown by P NPs, Glu-APD can provide the NPs with optional endocytosis pathways (i.e. clathrin-mediated, caveolae-mediated and micropinocytosis pathways), with the involvement of oligopeptide transporters and amino acid transporters, subsequently leading to a broadened intracellular trafficking route via the endoplasmic reticulum (ER) and Golgi apparatus. Furthermore, l-glutamic acid (l-Glu), a natural nutrient, could specifically facilitate the exocytosis of Glu-APD NPs, indicating an amino-acid-associated intracellular trafficking. Oral administration of insulin-loaded Glu-APD NPs could also achieve a good hypoglycemic effect with a relative bioavailability of 10.04%, which is 1.89-fold higher than that of P NPs and 5.20-fold higher than insulin solution. Safety evaluations further verified the biocompatibility of Glu-APD NPs and the related materials. The results confirmed the feasibility of introducing Glu-APD to NPs for improving the oral delivery of insulin. With the surface features of dendritic peptide, Glu-APD could facilitate oligopeptide/amino-acid-associated transport of the related NPs, which might be considered as an advantage under physiological conditions. This work might also be considered as a valid reference for the construction of highly efficient oral delivery systems.

Multifunctional Nanoparticles Enable Efficient Oral Delivery of Biomacromolecules via Improving Payload Stability and Regulating the Transcytosis Pathway.

In oral delivery of biomacromolecules, ligand-modified nanoparticles (NPs) have emerged as a promising tool to improve the epithelial uptake of the loaded protein/peptide. Unfortunately, the stability and the transport mechanisms of the biotherapeutics during the intracellular transportation still remained unclear, leading to the poor transepithelial efficiency. Additionally, developing novel approaches to simultaneously monitor the payload bioactivity during the transport processes is veritably benefit for keeping their bioactivity. In the present study, EGP peptide (KRKKKGKGLGKKRDPCLRKYK), a ligand with high affinity to heparan sulfate proteoglycans (HSPGs), was found remarkably increasing the cellular uptake (4.5-fold) and also surprisingly achieving high transcytosis efficiency (4.2-fold) of poly(lactide- co-glycolide) NPs on Caco-2 cell monolayer. Compared with unmodified NPs (C NPs), EGP modified NPs (EGP NPs) exhibited more desirable colloidal stability within epithelia. In the subsequent study, the bioactivity of encapsulated insulin during the cellular transportation was innovatively monitored by a glucose consumption assay. Inspiringly, EGP NPs could mostly retain the bioactivity of loaded insulin whereas insulin from INS-C NPs was significantly degraded. Then the detailed mechanism study revealed that the binding of EGP to HSPGs played a vital role on NP transportation. Unlike C NPs being delivered in the endo/lysosomal pathway, EGP NPs were involved in caveolae-mediated transport, which contributes to the efficient avoidance of the lysosomal entrapment and sequentially facilitates the direct apical-to-basolateral transcytosis. The enhanced absorption of EGP NPs was confirmed in in situ intestinal loop models. Most importantly, oral administrated INS-EGP NPs generated a strong hypoglycemic response on diabetic rats with 10.2-fold and 2.6-fold increase in bioavailability compared with free insulin and INS-C NPs, respectively. The work provided an innovative strategy to monitor the payload bioactivity during the transport processes and proposed a novel aspect to increase oral bioavailability of biomacromolecules via improving payload stability and regulating the transcytosis pathway of nanocarriers.

Biomimetic Viruslike and Charge Reversible Nanoparticles to Sequentially Overcome Mucus and Epithelial Barriers for Oral Insulin Delivery.

Nanoparticles (NPs) for oral delivery of peptide/protein drugs are largely limited due to the coexistence of intestinal mucus and epithelial barriers. Sequentially overcoming these two barriers is intractable for a single nanovehicle due to the requirements of different or even contradictory surface properties of NPs. To solve this dilemma, a mucus-penetrating virus-inspired biomimetic NP with charge reversal ability (P-R8-Pho NPs) was developed by densely coating poly(lactic- co-glycolic acid) NPs with cationic octa-arginine (R8) peptide and specific anionic phosphoserine (Pho). The small size (81.81 nm) and viruslike neutral charged surface (-2.39 mV) of the biomimetic NPs achieved rapid mucus penetration, which was almost equal to that of the conventional PEGylated mucus-penetrating nanoparticles. The hydrolysis of surface-anchored anionic Pho was achieved by intestinal alkaline phosphatase, which led to the turnover of ζ potential to positive (+7.37 mV). This timely charge reversal behavior also exposed cationic R8 peptide and induced efficient cell-penetrating peptide (CPP)-mediated cellular uptake and transepithelial transport on Caco-2/E12 cocultured cell model. What's more, P-R8-Pho NPs showed excellent stability in simulated gastrointestinal conditions and enhanced absorption in intestine in vivo. Finally, oral administration of insulin-loaded P-R8-Pho NPs enabled to induce a preferable hypoglycemic effect and a 1.9-fold higher oral bioavailability was achieved compared with single CPP-modified P-R8 NPs on diabetic rats. The combinative application of biomimetic mucus-penetrating strategy and enzyme-responsive charge reversal strategy in a single nanovehicle could sequentially overcome mucus and epithelial barriers, thus showing great potential for the oral peptide/protein delivery.

Novel Solid Lipid Nanoparticle with Endosomal Escape Function for Oral Delivery of Insulin.

Although nanoparticles (NPs) have been demonstrated as promising tools for improving oral absorption of biotherapeutics, most of them still have very limited oral bioavailability. Lyso-endosomal degradation in epithelial cells is one of the important but often-neglected physiological barriers, limiting the transport of cargoes across the intestinal epithelium. We herein reported a solid lipid nanoparticle (SLN) platform with a unique feature of endosomal escape for oral protein drug delivery. The SLNs consisted of a solid-lipid shell, which contained an endosomal escape agent (GLFEAIEGFIENGWEGMIDGWYG, HA2), and an aqueous core that is loaded with insulin (INS HA2-O-SLNs). SLNs without and with the HA2 peptide in the aqueous core (INS SLNs and INS HA2-W-SLNs, respectively) were used as the control groups. Our study showed that INS HA2-O-SLNs effectively facilitated the escape of the loaded insulin from the acidic endosomes, which preserved the biological activity of insulin to a greater extent during the intracellular transport. The spatial location of the HA2 peptide was demonstrated to determine the endosomal escape efficiency. As demonstrated in the intracellular trafficking of SLNs, INS HA2-O-SLNs displayed much less distribution in late endosomes and lysosomes. Meanwhile, insulin in INS HA2-O-SLNs exhibited the highest transepithelial permeation efficiency, with 2.19 and 1.72 folds higher accumulated amount in the basolateral side as compared to that in INS SLNs and INS HA2-W-SLNs. In addition, insulin from INS HA2-O-SLNs exhibited the highest insulin permeation in various regions of small intestines. INS HA2-O-SLNs generated an excellent hypoglycemic response following oral administration in diabetic rats. Thus, such functional SLNs demonstrated a great potency for oral delivery of peptide/protein drugs.

The combination of endolysosomal escape and basolateral stimulation to overcome the difficulties of "easy uptake hard transcytosis" of ligand-modified nanoparticles in oral drug delivery.

Ligand-modified nanoparticles (NPs) are an effective tool to increase the endocytosis efficiency of drugs, but these functionalized NPs face the drawback of "easy uptake hard transcytosis" in the oral delivery of proteins and peptides. Adversely, the resulting deficiency in transcytosis has not attracted much attention. Herein, NPs modified with the low-density lipoprotein receptor (LDLR) ligand NH2-C6-[cMPRLRGC]c-NH2, i.e., peptide-22 (P22NPs) were fabricated to investigate strategies related to the enhancement of transcytosis. By systematically studying the intracellular trafficking of NPs, it was found that reduced transcytosis might be associated with the entrapment of P22NPs in endosomes or lysosomes and limited basolateral exocytosis. On this basis, the prevention of the endolysosomal entrapment of NPs and the acceleration of basolateral exocytosis should be considered as strategies to enhance the transcytosis of NPs. By screening chemicals that could help the endosomal/lysosomal escape of chemicals related to LDLR-mediated transcytosis, it was shown that hemagglutinin-2 (HA2) and metformin had higher abilities to enhance the exocytosis of P22NPs. The transcytosis efficiencies of insulin loaded in P22NPs were also investigated, and a 3.2-fold increase in transcytosis was observed in comparison with free insulin. The transcytosis efficiencies of insulin could be further increased by the addition of metformin or HA2 (3.6-fold or 4.1-fold higher than that of free insulin). Inspiringly, the simultaneous addition of the abovementioned two chemicals led to the highest transcytosis efficiency of insulin, which was up to 5.1-fold higher than that of free insulin. These results demonstrated that endolysosomal entrapment and basolateral exocytosis are two of the most important limiting steps for the "easy uptake hard transcytosis" of orally administered ligand-modified NPs. Moreover, our work provides a new point of view for the design of novel oral drug delivery systems.

Enhanced Oral Delivery of Protein Drugs Using Zwitterion-Functionalized Nanoparticles to Overcome both the Diffusion and Absorption Barriers.

Oral delivery of protein drugs based on nanoparticulate delivery system requires permeation of the nanoparticles through the mucus layer and subsequent absorption via epithelial cells. However, overcoming these two barriers requires very different or even contradictory surface properties of the nanocarriers, which greatly limits the oral bioavailability of macromolecular drugs. Here we report a simple zwitterions-based nanoparticle (NP) delivery platform, which showed a great potency in simultaneously overcoming both the mucus and epithelium barriers. The dense and hydrophilic coating of zwitterions endows the NPs with excellent mucus penetrating ability. Moreover, the zwitterions-based NPs also possessed excellent affinity with epithelial cells, which significantly improved (4.5-fold) the cellular uptake of DLPC NPs, compared to PEGylated NPs. Our results also indicated that this affinity was due to the interaction between zwitterions and the cell surface transporter PEPT1. Moreover, the developed NPs loaded with insulin could induce a prominent hypoglycemic response in diabetic rats following oral administration. These results suggest that zwitterions-based NPs might provide a new perspective for oral delivery of protein therapeutics.