Long-Acting Heterodimeric Paclitaxel-Idebenone Prodrug-Based Nanomedicine Promotes Functional Recovery after Spinal Cord Injury.

After spinal cord injury (SCI), successive systemic administration of microtubule-stabilizing agents has been shown to promote axon regeneration. However, this approach is limited by poor drug bioavailability, especially given the rapid restoration of the blood-spinal cord barrier. There is a pressing need for long-acting formulations of microtubule-stabilizing agents in treating SCI. Here, we conjugated the antioxidant idebenone with microtubule-stabilizing paclitaxel to create a heterodimeric paclitaxel-idebenone prodrug via an acid-activatable, self-immolative ketal linker and then fabricated it into chondroitin sulfate proteoglycan-binding nanomedicine, enabling drug retention within the spinal cord for at least 2 weeks and notable enhancement in hindlimb motor function and axon regeneration after a single intraspinal administration. Additional investigations uncovered that idebenone can suppress the activation of microglia and neuronal ferroptosis, thereby amplifying the therapeutic effect of paclitaxel. This prodrug-based nanomedicine simultaneously accomplishes neuroprotection and axon regeneration, offering a promising therapeutic strategy for SCI.

Type I Collagen-Adhesive and ROS-Scavenging Nanoreactors Enhanced Retinal Ganglion Cell Survival in an Experimental Optic Nerve Crush Model.

Traumatic optic neuropathy (TON) is a severe condition characterized by retinal ganglion cell (RGC) death, often leading to irreversible vision loss, and the death of RGCs is closely associated with oxidative stress. Unfortunately, effective treatment options for TON are lacking. To address this, catalase (CAT) is encapsulated in a tannic acid (TA)/poly(ethylenimine)-crosslinked hollow nanoreactor (CAT@PTP), which exhibited enhanced anchoring in the retina due to TA-collagen adhesion. The antioxidative activity of both CAT and TA synergistically eliminated reactive oxygen species (ROS) to save RGCs in the retina, thereby treating TON. In vitro experiments demonstrated that the nanoreactors preserve the enzymatic activity of CAT and exhibit high adhesion to type I collagen. The combination of CAT and TA-based nanoreactors enhanced ROS elimination while maintaining high biocompatibility. In an optic nerve crush rat model, CAT@PTP is effectively anchored to the retina via TA-collagen adhesion after a single vitreous injection, and RGCs are significantly preserved without adverse events. CAT@PTP exhibited a protective effect on retinal function. Given the abundance of collagen that exists in ocular tissues, these findings may contribute to the further application of this multifunctional nanoreactor in ocular diseases to improve therapeutic efficacy and reduce adverse effects.

Stromal Homeostasis-Restoring Nanomedicine Enhances Pancreatic Cancer Chemotherapy.

The desmoplastic stroma imposes a fatal physical delivery barrier in pancreatic ductal adenocarcinoma (PDAC) therapy. Deconstructing the stroma components hence predominates in stroma-targeting approaches, but conflicting outcomes have sometimes occurred due to the multifaceted nature of the stroma. Here, we constructed two sub-20-nm nanomedicines based on a so-called "next-wave" antifibrotic halofuginone (HF) and the tumoricidal paclitaxel (PTX) for enhanced PDAC chemotherapy. This was achieved by coassembling methoxy poly(ethylene glycol)-b-poly(caprolactone) with ketal-linked HF- and PTX-derived prodrugs. HF nanomedicine and PTX nanomedicine had excellent prodrug-nanocarrier compatibility and exhibited greatly improved pharmacokinetic profiles and high tumor accumulation. HF nanomedicine pretreatment restored stromal homeostasis and considerably facilitated the distribution of PTX nanomedicine and its penetration into carcinoma cells, leading to positive modulation of the infiltration of cytotoxic T cells and significant regression of tumor growth in two PDAC models. Our nanomedicine-based stromal remodeling strategy appears promising for treating desmoplastic malignancies.

Microcrystals of Ketal-Linked Paliperidone Prodrugs for Long-Acting Antipsychotics.

Intramuscularly injectable long-acting prodrug-based microcrystals (MCs) are of particular interest for chronic disease management. Nevertheless, current prevalently used linkers degraded by enzymes have the potential drawback of substantial differences in enzyme levels between individuals. Here, we reported the synthesis of a stearyl-modified paliperidone prodrug (SKP) with an acid-sensitive ketal linker for developing long-acting MC antipsychotics. SKP-MCs of three different sizes were prepared and systematically examined. We found that paliperidone exposure in SKP-MC-treated rats was prolonged compared with that in rats treated with the commercial antipsychotic Invega Sustenna and that the drug release rate decreased with increasing MC size. In inflammation-inhibition-model rats, paliperidone release from the SKP-MCs was considerably decreased, indicating that the immune-mediated foreign-body response after intramuscular administration boosted paliperidone release. Our findings will provide valuable insights into in vivo drug release from prodrug-based MC formulations. The ketal-linked prodrug strategy might be a new solution for developing long-acting prodrug formulations of hydroxyl-group-bearing drugs.

Modular Acid-Activatable Acetone-Based Ketal-Linked Nanomedicine by Dexamethasone Prodrugs for Enhanced Anti-Rheumatoid Arthritis with Low Side Effects.

Given the physically encapsulated payloads with drug burst release and/or low drug loading, it is critical to initiate an innovative prodrug strategy to optimize the design of modular nanomedicines. Here, we designed modular pH-sensitive acetone-based ketal-linked prodrugs of dexamethasone (AKP-dexs) and formulated them as nanoparticles. We comprehensively studied the relationships between AKP-dex structure and properties, and we selected two types of AKP-dex-loaded nanoparticles for in vivo studies on the basis of their size, drug loading, and colloidal stability. In a collagen-induced arthritis rat model, these AKP-dex-loaded nanoparticles showed higher accumulation in inflamed joints and better therapeutic efficacy than free dexamethasone phosphate with less-severe side effects. AKP-dex-loaded nanoparticles may be useful for treating other inflammatory diseases and thus have great translational potential. Our findings represent an important step toward the development of practical applications for acetone-based ketal-linked prodrugs and are useful in the design of modular nanomedicines.

Dually Enzyme- and Acid-Triggered Self-Immolative Ketal Glycoside Nanoparticles for Effective Cancer Prodrug Monotherapy.

The use of glycoside prodrugs is a promising strategy for developing new targeted medicines for chemotherapy. However, the in vivo utility of such prodrugs is hindered by insufficient activation and the lack of convenient synthetic methods. We have developed an innovative strategy for synthesizing ketal glycoside prodrugs that are unique in being activated by a dual enzyme- and acid-triggered self-immolative mechanism. Amphiphilic glucosyl acetone-based ketal-linked etoposide glycoside prodrug isomers were synthesized and fabricated into excipient-free nanoparticles for effective cancer prodrug monotherapy. Hydrolysis of the glycosidic linkage or the ketal linkage triggered hydrolysis of the other linkage, which resulted in spontaneous self-immolative hydrolysis of the prodrugs. Nanoparticles of the prodrug isomer that was the most labile in a lysosome-mimicking environment displayed high intratumoral accumulation and strong antitumor activity in an A549 xenograft mouse model. Our strategy may be useful for the development of stimulus-responsive self-immolative prodrugs and their nanomedicines.

Acid-Triggered Release of Native Gemcitabine Conjugated in Polyketal Nanoparticles for Enhanced Anticancer Therapy.

Nucleoside analogue drugs are widely used in cancer therapy and antiviral therapy, while fast metabolism, drug resistance, and severe side effects significantly limit their clinical applications. To address these issues, a variety of ester- and amide-linked prodrugs and their nanoparticulate formulations have been devised. However, most of these prodrugs suffer from inefficient transformation to native drugs in tumor. Here, we report an approach to conjugate gemcitabine, a kind of anticancer nucleoside drug and widely used to treat cancers, to polyketal backbone via pH-sensitive ketal linkage, and prepared gemcitabine-containing polyketal prodrug nanoparticles with minimal drug release under physiological conditions and acid-triggerable release of native gemcitabine. Intracellular and intratumoral degradation of the pH-sensitive gemcitabine-containing polyketal prodrug and incorporation of gemcitabine into DNA were confirmed by confocal microscopy using EdU, an analogue of gemcitabine. One single intravenous injection of these gemcitabine-containing polyketal prodrug nanoparticles demonstrated notable anticancer efficacy in the A2780 ovarian xenograft tumor model with increased survival rate and good safety. Our approach can be adopted for other diol nucleoside analogues to synthesize pH-sensitive nucleoside-polyketal prodrugs for developing anticancer and antiviral formulations.

Interaction kinetics of peptide lipids-mediated gene delivery.

BACKGROUND: During the course of gene transfection, the interaction kinetics between liposomes and DNA is speculated to play very important role for blood stability, cellular uptake, DNA release and finally transfection efficiency. RESULTS: As cationic peptide liposomes exhibited great gene transfer activities both in vitro and in vivo, two peptide lipids, containing a tri-ornithine head (LOrn3) and a mono-ornithine head (LOrn1), were chosen to further clarify the process of liposome-mediated gene delivery in this study. The results show that the electrostatically-driven binding between DNA and liposomes reached nearly 100% at equilibrium, and high affinity of LOrn3 to DNA led to fast binding rate between them. The binding process between LOrn3 and DNA conformed to the kinetics equation: y = 1.663631 × exp (- 0.003427x) + 6.278163. Compared to liposome LOrn1, the liposome LOrn3/DNA lipoplex exhibited a faster and more uniform uptake in HeLa cells, as LOrn3 with a tri-ornithine peptide headgroup had a stronger interaction with the negatively charged cell membrane than LOrn1. The efficient endosomal escape of DNA from LOrn3 lipoplex was facilitated by the acidity in late endosomes, resulting in broken carbamate bonds, as well as the "proton sponge effect" of the lipid. CONCLUSIONS: The interaction kinetics is a key factor for DNA transfection efficiency. This work provided insights into peptide lipid-mediated DNA delivery that could guide the development of the next generation of delivery systems for gene therapeutics.

Synthesis of Poly(acyclic orthoester)s: Acid-Sensitive Biomaterials for Enhancing Immune Responses of Protein Vaccine.

While poly(acyclic orthoester)s (PAOEs) have many appealing features for drug delivery, their application is significantly hindered by a lack of facile synthetic methods. Reported here is a simple method for synthesizing acyclic diketene acetal monomers from diols and vinyl ether, and their polymerization with a diol to first synthesize PAOEs. The PAOEs rapidly hydrolyze at lysosomal pH. With the help of a cationic lipid, ovalbumin, a model vaccine antigen was efficiently loaded into PAOEs nanoparticles using a double emulsion method. These nanoparticles efficiently delivered ovalbumin into the cytosol of dendritic cells and demonstrated enhanced antigen presentation over poly(lactic-co-glycolic acid) (PLGA) nanoparticles. PAOEs are promising vehicles for intracellular delivery of biopharmaceuticals and could increase the utility of poly(orthoesters) in biomedical research.

Phototriggered Local Anesthesia.

We report a phototriggerable formulation enabling in vivo repeated and on-demand anesthesia with minimal toxicity. Gold nanorods (GNRs) that can convert near-infrared (NIR) light into heat were attached to liposomes (Lip-GNRs), enabling light-triggered phase transition of their lipid bilayers with a consequent release of payload. Lip-GNRs containing the site 1 sodium channel blocker tetrodotoxin and the α2-adrenergic agonist dexmedetomidine (Lip-GNR-TD) were injected subcutaneously in the rat footpad. Irradiation with an 808 nm continuous wave NIR laser produced on-demand and repeated infiltration anesthesia in the rat footpad in proportion to the irradiance, with minimal toxicity. The ability to achieve on-demand and repeated local anesthesia could be very beneficial in the management of pain.

Extended Release of Native Drug Conjugated in Polyketal Microparticles.

Polyketals, which can be biodegradable, have good biocompatibility, and are pH-sensitive, could have broad applicability in drug delivery and other biomedical applications. However, facile synthesis of high molecular weight polyketals is challenging, and short durations of drug release from polyketal particulate formulations limit their application in drug delivery. Here we report the synthesis of a di-isopropenyl ether monomer and its use to synthesize high molecular weight estradiol-polyketal conjugates by addition polymerization. Microparticles were prepared from the estradiol-polyketal conjugate, where estradiol was incorporated into the polymer backbone. The particles had high drug loading and significantly prolonged drug release. Release of estradiol from the drug-polyketal conjugate microparticles was acid-responsive, as evidenced by faster drug release at low pH and with co-incorporation of PLGA. Tissue reaction to the microparticles was benign in vivo. Polyketal drug conjugates are promising candidates for long-acting drug delivery systems to treat chronic diseases.

Nanoparticles with Precise Ratiometric Co-Loading and Co-Delivery of Gemcitabine Monophosphate and Cisplatin for Treatment of Bladder Cancer.

Combination chemotherapy is a common practice in clinical management of malignancy. Synergistic therapeutic outcome is only achieved when tumor cells are exposed to cells in an optimal ratio. However, due to diverse physicochemical properties of drugs, no free drug cocktails or nanomaterials are capable of co-loading and co-delivering drugs at an optimal ratio. Herein, we develop a novel nano-platform with precise ratiometric co-loading and co-delivery of two hydrophilic drugs for synergistic anti-tumor effects. Based on previous work, we utilize a solvent displacement method to ratiometrically load dioleoyl phosphatidic acid (DOPA)-gemcitabine monophosphate and DOPA coated cisplatin-precipitate nanocores into the same PLGA NP. These cores are designed to have similar hydrophobic surface properties. GMP and cisplatin are engineered into PLGA NP at an optimal synergistic ratio (5:1, mol:mol) with over 70% encapsulation efficiency and were ratiometrically taken up by tumor cells in vitro and in vivo. These PLGA NP exhibit synergistic anti-cancer effects in a stroma-rich bladder tumor model. A single injection of dual drugs in PLGA NP can significantly inhibit tumor growth. This nanomaterial-system solves problems related to ratiometric co-loading and co-delivery of different hydrophilic moieties and provides possibilities for co-loading hydrophilic drugs with hydrophobic drugs for combination therapy.

Intravenous delivery of siRNA targeting CD47 effectively inhibits melanoma tumor growth and lung metastasis.

CD47 is a "self marker" that is usually overexpressed on the surface of cancer cells to enable them to escape immunosurveillance. Recognition of CD47 by its receptor, signal regulatory protein α (SIRPα), which is expressed in the macrophages, inhibits phagocytic destruction of cancer cells by the macrophages. In this study, we have first shown that clinical isolates of human melanoma significantly upregulate CD47, possibly as a mechanism to defend themselves against the macrophages. We then exploited RNA interference (RNAi) technology to test the hypothesis that knocking down CD47 in the tumor cells will render them targets for macrophage destruction; hence, creating a novel anti-cancer therapy. Anti-CD47 siRNA was encapsulated in a liposome-protamine-hyaluronic acid (LPH) nanoparticle (NP) formulation to address the challenge of targeted delivery of siRNA-based therapeutics in vivo. Efficient silencing of CD47 in tumor tissues with systemic administration of LPH(CD47) also significantly inhibited the growth of melanoma tumors. In a lung metastasis model, LPH(CD47) efficiently inhibited lung metastasis to about 27% of the untreated control. Moreover, no hematopoietic toxicity was observed in the animals that received multiple doses of LPH(CD47). Our findings indicate CD47 as a potential prognostic marker for melanoma development as well as a target for therapeutic intervention with RNAi-based nanomedicines.

Tumor-penetrating iRGD facilitates penetration of poly(floxuridine-ketal)-based nanomedicine for enhanced pancreatic cancer therapy.

Efficient intratumoral penetration is essential for nanomedicine to eradicate pancreatic tumors. Although nanomedicine can enter the perivascular space of pancreatic tumors, their access to distal tumor cells, aloof from the vessels, remains a formidable challenge. Here, we synthesized an acid-activatable macromolecular prodrug of floxuridine (FUDR)-poly(FUDR-ketal), engineered a micellar nanomedicine of FUDR, and intravenously co-administered the nanomedicine with the tumor-penetrating peptide iRGD for enhanced treatment of pancreatic tumor. A FUDR-derived mono-isopropenyl ether was synthesized and underwent self-addition polymerization to afford the hydrophobic poly(FUDR-ketal), which was subsequently co-assembled with amphiphilic DSPE-mPEG into the micellar nanomedicine with size of 12 nm and drug content of 56.8 wt% using nanoprecipitation technique. The acetone-based ketal-linked poly(FUDR-ketal) was triggered by acid to release FUDR to inhibit cell proliferation. In an orthotopic pancreatic tumor model derived from KPC (KrasLSL-G12D/+; Trp53LSL-R172H/+; Pdx1-Cre) cells that overexpress neuropilin-1 (NRP-1) receptor, iRGD improved penetration of FUDR nanomedicine into tumor parenchyma and potentiated the therapeutic efficacy. Our nanoplatform, along with iRGD, thus appears to be promising for efficient penetration and activation of acid-responsive nanomedicines for enhanced pancreatic cancer therapy.

Enhanced bone regeneration by osteoinductive and angiogenic zein/whitlockite composite scaffolds loaded with levofloxacin.

Promoting angiogenesis following biomaterial implantation is essential to bone tissue regeneration. Herein, the composite scaffolds composed of zein, whitlockite (WH), and levofloxacin (LEVO) were fabricated to augment bone repair by facilitating osteogenesis and angiogenesis. First, three-dimensional composite scaffolds containing zein and WH were prepared using the salt-leaching method. Then, as a model antibiotic drug, the LEVO was loaded into zein/WH scaffolds. Moreover, the addition of WH enhanced the adhesion, differentiation, and mineralization of osteoblasts. The zein/WH/LEVO composite scaffolds not only had significant osteoinductivity but also showed excellent antibacterial properties. The prepared composite scaffolds were then implanted into a calvarial defect model to evaluate their osteogenic induction effects in vivo. Micro-CT observation and histological analysis indicate that the scaffolds can accelerate bone regeneration with the contribution of endogenous cytokines. Based on amounts of data in vitro and in vivo, the scaffolds present profound effects on improving bone regeneration, especially for the favorable osteogenic, intensive angiogenic, and alleviated inflammation abilities. The results showed that the synthesized scaffolds could be a potential material for bone tissue engineering.

Drug content on anticancer efficacy of self-assembling ketal-linked dextran-paclitaxel conjugates.

Although polymer-drug conjugates (PDCs) show great promise as versatile drug delivery systems, no antitumor PDCs based on small-molecule drugs are currently on the market, partly because of the lack of validated design principles for PDCs. High drug content is thought to be essential for devising highly efficacious PDCs based on poorly soluble antitumor drugs, but this has not been well validated. Therefore, revisiting the relationship between drug content and PDC performance is vital. In this study, we synthesized four dextran-paclitaxel (PTX) conjugates (designated as DKPs) with different drug contents by linking dextran and PTX via an acid-responsive ketal, and we used the conjugates to construct self-assembled DKP nanoparticles (NPs) for antitumor therapy. We focused on how PTX content influenced the hydrolysis kinetics, cytotoxicity, cellular uptake and intracellular hydrolysis, pharmacokinetics, biodistribution, and antitumor efficacies of the DKP NPs. We found that DKP NPs with lower PTX content showed accelerated drug release and increased tumor accumulation, and consequently enhanced antitumor efficacy. In 4T1-Luc and Panc02-Luc cancer models, the NPs showed considerably improved therapeutic efficacy than the micellar formulation of PTX that is currently in clinical use. Our results indicate that DKP NPs with lower PTX content possess greater antitumor potential, and our findings offer new insights for the connection of drug content-formulation-bioactivity relationship in the rational design of PDC prodrugs.

Fine-tuning the sequential drug release of nano-formulated mutual prodrugs dictates the combination effects.

The maintenance of robust ratiometric loading of dual therapeutic agents and fine-tuning release kinetics for consistent in vitro and in vivo optimization of combination effects is vital for discovering new anticancer drug combinations and remains challenging. Smart nanomedicine strategies have been investigated for this purpose, but most of the reported strategies focus either on ratiometric delivery or on unimodal sequential release of the two different agents, which hampers effective optimization of combination effects. Herein we report a sequential drug release system based on nanoformulated mutual prodrugs constructed by the formation of ketal linkages with different acid sensitivities, thus enabling the acid-triggered release of two anticancer drugs, paclitaxel and gemcitabine, in various sequences. We found that in several cell lines, the sequence of drug release substantially affected the combination effects; specifically, in A549 cells, time-staggered release profiles showed enhanced synergistic effects relative to those of a simultaneous release profile. Moreover, in vivo assessment of the antitumor efficacy of the nanoformulations in A549 xenograft models indicated that the best therapeutic effects were obtained with time-staggered release profiles, which was consistent with the in vitro results. Our strategy for precisely controlled sequential drug release can be expected to facilitate the screening of optimal drug combinations and maximize combination effects both in vitro and in vivo.

The Yin and Yang of the protein corona on the delivery journey of nanoparticles.

Nanoparticles-based drug delivery systems have attracted significant attention in biomedical fields because they can deliver loaded cargoes to the target site in a controlled manner. However, tremendous challenges must still be overcome to reach the expected targeting and therapeutic efficacy in vivo. These challenges mainly arise because the interaction between nanoparticles and biological systems is complex and dynamic and is influenced by the physicochemical properties of the nanoparticles and the heterogeneity of biological systems. Importantly, once the nanoparticles are injected into the blood, a protein corona will inevitably form on the surface. The protein corona creates a new biological identity which plays a vital role in mediating the bio-nano interaction and determining the ultimate results. Thus, it is essential to understand how the protein corona affects the delivery journey of nanoparticles in vivo and what we can do to exploit the protein corona for better delivery efficiency. In this review, we first summarize the fundamental impact of the protein corona on the delivery journey of nanoparticles. Next, we emphasize the strategies that have been developed for tailoring and exploiting the protein corona to improve the transportation behavior of nanoparticles in vivo. Finally, we highlight what we need to do as a next step towards better understanding and exploitation of the protein corona. We hope these insights into the "Yin and Yang" effect of the protein corona will have profound implications for understanding the role of the protein corona in a wide range of nanoparticles.

Elimination pathways of systemically delivered siRNA.

The elimination process of systemically administered small interfering RNA (siRNA) was investigated by using siRNA labeled with an infrared fluorescent dye. A novel siRNA elimination pathway was identified. In this pathway, liver-enriched siRNA is secreted into the gallbladder and then emptied into the intestine. Blocking this pathway resulted in the absence of siRNA fluorescence within the intestine, with greatly enhanced siRNA accumulation in liver and gallbladder at the same time. Furthermore, we demonstrated that delivery carriers play an essential role in siRNA distribution and elimination, highlighting their importance in siRNA therapeutics.

Long-acting acid-sensitive ketal-linked dexamethasone microcrystals for treating experimental autoimmune uveitis.

Corticosteroids have for some time been used as first-line drugs for the topical treatment of noninfectious uveitis, but poor ocular bioavailability and the rapid clearance of eye drops necessitate frequent dosing, reducing patient compliance. In this study, we used an acid-sensitive stearoxyl-ketal-dexamethasone pro-drug microcrystals (SKD MCs), which is consistently safe and effective in the control of uveitis inflammation in rats. We used a rat model of experimental autoimmune uveitis (EAU) to evaluate the effects of SKD MCs in terms of clinical manifestations, molecular biology, pathological histology, and visual electrophysiology compared to dexamethasone sodium phosphate injection or phosphate-buffered saline. SKD MCs significantly reduced inflammation in EAU, improved the ability to suppress inflammatory cytokines and to protect retinal function, and significantly reduced retinal microglia activation, with no increase in intraocular pressure throughout the treatment. Our results indicate that the SKD MCs formulation holds promise as a new strategy for the treatment of noninfectious uveitis and potentially other ocular inflammatory diseases.