Recent Progress in Polyion Complex Nanoparticles with Enhanced Stability for Drug Delivery.

Polyion complex (PIC) nanoparticles, including PIC micelles and PICsomes, are typically composed of poly(ethylene glycol) block copolymers coupled with oppositely charged polyelectrolytes or therapeutic agents via electrostatic interaction. Due to a simple and rapid preparation process with high drug-loading efficiency, PIC nanoparticles are beneficial to maintaining the chemical integrity and high biological activity of the loaded drugs. However, the stability of PIC nanoparticles can be disrupted in high-ionic-strength solutions because electrostatic interaction is the DRIVING force; these disruptions can thus impair drug delivery. Herein, we summarize the advances in the use of PIC nanoparticles for delivery of charged drugs, focusing on the different chemical and physical strategies employed to enhance their stability, including enhancing the charge density, crosslinking, increasing hydrophobic interactions, forming hydrogen bonds, and the development of PIC-based gels. In particular, we describe the use of PIC nanoparticles to load peptide antibiotics targeting antibiotic-resistant and biofilm-related diseases and the use of nanoparticles that load chemotherapeutics and gaseous donors for cancer treatment. Furthermore, the application of PIC nanoparticles as magnetic resonance imaging contrast agents is summarized for the first time. Therefore, this review is of great significance for advances in the use of polymeric nanoparticles for functional drug delivery.

Early protein delivery in critically ill patients with acute kidney injury: post hoc analysis of a multicenter cluster-randomized controlled trial.

Background: There is controversy over the optimal early protein delivery in critically ill patients with acute kidney injury (AKI). This study aims to evaluate whether the association between early protein delivery and 28-day mortality was impacted by the presence of AKI in critically ill patients. Methods: This is a post hoc analysis of data from a multicenter cluster-randomised controlled trial enrolling newly admitted critically ill patients (n = 2772). Participants without chronic kidney disease and with complete data concerning baseline renal function were included in this study. The primary outcome was 28-day mortality. Cox proportional hazards models were used to analyze the association between early protein delivery, reflected by mean protein delivery from day 3-5 after enrollment, 28-day mortality and whether baseline AKI stages interacted with this association. Results: Overall, 2552 patients were included, among whom 567 (22.2%) had AKI at enrollment (111 stage I, 87 stage II, 369 stage III). Mean early protein delivery was 0.60 ± 0.38 g/kg/day among the study patients. In the overall study cohort, each 0.1 g/kg/day increase in protein delivery was associated with a 5% reduction in 28-day mortality[hazard ratio (HR) = 0.95; 95% confidence interval (CI) 0.92-0.98, p < 0.001]. The association between early protein delivery and 28-day mortality significantly interacted with baseline AKI stages (adjusted interaction p = 0.028). Each 0.1 g/kg/day increase in early protein delivery was associated with a 4% reduction in 28-day mortality (HR = 0.96; 95%CI 0.92-0.99, p = 0.011) among patients without AKI and 9% (HR = 0.91; 95%CI 0.84-0.99, p = 0.021) among those with AKI stage III. However, such associations cannot be observed among patients with AKI stages I and II. Conclusions: Increased early protein delivery (up to close to the guideline recommendation) was associated with reduced 28-day mortality in critically ill patients without AKI and with AKI stage III, but not in those with AKI stage I or II.

An approach for the intracellular delivery of IgG via enzymatic ligation with a cell-permeable attenuated cationic amphiphilic lytic peptide.

Achieving effective intracellular delivery of therapeutic molecules such as antibodies (IgG) is a challenge in biomedical research and pharmaceutical development. Conjugation of IgG with a cell-penetrating peptide is a rational approach. Here, not only the efficacy of the conjugates in internalizing into cells, but also the physicochemical property of the conjugates allowing their solubilized states in solution without forming aggregates are critical. In this study, we have shown that the first requirement can be addressed using a cell-permeable attenuated cationic amphiphilic lytic (CP-ACAL) peptide, L17ER4. The second requirement can be addressed by ligation of IgG to L17ER4 using sortase A, where the use of a linker of appropriate chain length is also important. For evaluation, the intracellular delivery efficacy was studied using conjugate structures with different orientations and conjugation modes of L17ER4 in ligation to a model protein, green fluorescent protein fused to a nuclear localization signal (NLS-EGFP). The effect of tetraarginine positioning in the L17ER4 sequence was also investigated. Following these studies, an optimized peptide sequence containing L17ER4 was ligated to an anti-green fluorescent protein (GFP) IgG bearing a sortase A recognition sequence. Treatment of the cells with the conjugate of anti-GFP IgG and L17ER4 resulted in a high efficiency of cytosolic translocation of the conjugate and the binding to the target protein in the cell without significant aggregate formation. The feasibility of the d-form of L17ER4 as a CP-ACAL was also confirmed.

Specific targeting of cancer vaccines to antigen-presenting cells via an endogenous TLR2/6 ligand derived from cysteinyl-tRNA synthetase 1.

Cancer vaccines have been developed as a promising way to boost cancer immunity. However, their clinical potency is often limited due to the imprecise delivery of tumor antigens. To overcome this problem, we conjugated an endogenous Toll-like receptor (TLR)2/6 ligand, UNE-C1, to human papilloma virus type 16 (HPV-16)-derived peptide antigen, E7, and found that the UNE-C1-conjugated cancer vaccine (UCV) showed significantly enhanced antitumor activity in vivo compared with the noncovalent combination of UNE-C1 and E7. The combination of UCV with PD-1 blockades further augmented its therapeutic efficacy. Specifically, the conjugation of UNE-C1 to E7 enhanced its retention in inguinal draining lymph nodes, the specific delivery to dendritic cells and E7 antigen-specific T cell responses, and antitumor efficacy in vivo compared with the noncovalent combination of the two peptides. These findings suggest the potential of UNE-C1 derived from human cysteinyl-tRNA synthetase 1 as a unique vehicle for the specific delivery of cancer antigens to antigen-presenting cells via TLR2/6 for the improvement of cancer vaccines.

Finding ways into the cytosol: Peptide-mediated approaches for delivering proteins into cells.

The delivery of functional proteins, including antibodies, into cells opens up many opportunities to regulate cellular events, with significant implications for studies in chemical biology and therapeutics. The inside of cells is isolated from the outside by the cell membrane. The hydrophilic nature of proteins prevents direct permeation of proteins through the cell membrane by passive diffusion. Therefore, delivery routes using endocytic uptake followed by endosomal escape have been explored. Alternatively, delivery concepts using transient permeabilization of cell membranes or effective promotion of endocytic uptake and endosomal escape using modified membrane-lytic peptides have been reported in recent years. Non-canonical protein delivery concepts, such as the use of liquid droplets or coacervates, have also been proposed. This review highlights some of the topics in peptide-mediated intracellular protein delivery.

Enhancing CRISPR prime editing by reducing misfolded pegRNA interactions.

CRISPR prime editing (PE) requires a Cas9 nickase-reverse transcriptase fusion protein (known as PE2) and a prime editing guide RNA (pegRNA), an extended version of a standard guide RNA (gRNA) that both specifies the intended target genomic sequence and encodes the desired genetic edit. Here, we show that sequence complementarity between the 5' and the 3' regions of a pegRNA can negatively impact its ability to complex with Cas9, thereby potentially reducing PE efficiency. We demonstrate this limitation can be overcome by a simple pegRNA refolding procedure, which improved ribonucleoprotein-mediated PE efficiencies in zebrafish embryos by up to nearly 25-fold. Further gains in PE efficiencies of as much as sixfold could also be achieved by introducing point mutations designed to disrupt internal interactions within the pegRNA. Our work defines simple strategies that can be implemented to improve the efficiency of PE.

Targeted protein delivery based on stimuli-triggered nanomedicine.

Protein-based drugs have shown unique advantages to treat various diseases in recent years. However, most protein therapeutics in clinical use are limited to extracellular targets with low delivery efficiency. To realize targeted protein delivery, a series of stimuli-triggered nanoparticle formulations have been developed to improve delivery efficiency and reduce off-target release. These smart nanoparticles are designed to release cargo proteins in response to either internal or external stimuli at pathological tissues. In this way, varieties of protein-based drugs including antibodies, enzymes, and pro-apoptotic proteins can be effectively delivered to desired sites for the treatment of cancer, inflammation, metabolic diseases, and so on with minimal side effects. In this review, recent advances in the design of stimuli-triggered nanomedicine for targeted protein delivery in different biomedical applications will be discussed. A deeper understanding of these emerging strategies helps develop more efficient protein delivery systems for clinical use in the future.

Rational design of diblock copolymer enables efficient cytosolic protein delivery.

Polymer-mediated cytosolic protein delivery demonstrates a promising strategy for the development of protein therapeutics. Here, we propose a new designed diblock copolymer which realizes efficient cytosolic protein delivery both in vitro and in vivo. The polymer contains one protein-binding block composed of phenylboronic acid (PBA) and N-(3-dimethylaminopropyl) (DMAP) pendant units for protein binding and endosomal escape, respectively, followed by the response to ATP enriched in the cytosol which triggers the protein release. The other block is PEG designed to improve particle size control and circulation in vivo. By optimizing the block composition, sequence and length of the copolymer, the optimal one (BP20) was identified with the binding block containing 20 units of both PBA and DMAP, randomly distributed along the chain. When mixed with proteins, the BP20 forms stable nanoparticles and mediates efficient cytosolic delivery of a wide range of proteins including enzymes, toxic proteins and CRISPR/Cas9 ribonucleoproteins (RNP), to various cell lines. The PEG block, especially when further modified with folic acid (FA), enables tumor-targeted delivery of Saporin in vivo, which significantly suppresses the tumor growth. Our results shall inspire the design of novel polymeric vehicles with robust capability for cytosolic protein delivery, which holds great potential for both biological research and therapeutic applications.

Carbon Nanotube-Mediated Delivery of PTEN Variants: In Vitro Antitumor Activity in Breast Cancer Cells.

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a crucial tumor suppressor protein with frequent mutations and alterations. Although protein therapeutics are already integral to numerous medical fields, their potential remains nascent. This study aimed to investigate the impact of stable, unphosphorylated recombinant human full-length PTEN and its truncated variants, regarding their tumor suppression activity with multiwalled-carbon nanotubes (MW-CNTs) as vehicles for their delivery in breast cancer cells (T-47D, ZR-75-1, and MCF-7). The cloning, overexpression, and purification of PTEN variants were achieved from E. coli, followed by successful binding to CNTs. Cell incubation with protein-functionalized CNTs revealed that the full-length PTEN-CNTs significantly inhibited cancer cell growth and stimulated apoptosis in ZR-75-1 and MCF-7 cells, while truncated PTEN fragments on CNTs had a lesser effect. The N-terminal fragment, despite possessing the active site, did not have the same effect as the full length PTEN, emphasizing the necessity of interaction with the C2 domain in the C-terminal tail. Our findings highlight the efficacy of full-length PTEN in inhibiting cancer growth and inducing apoptosis through the alteration of the expression levels of key apoptotic markers. In addition, the utilization of carbon nanotubes as a potent PTEN protein delivery system provides valuable insights for future applications in in vivo models and clinical studies.

Polymers of functionalized diaminopropionic acid are efficient mediators of active exogenous enzyme delivery into cells.

Delivery of active protein especially enzyme is one of the major therapeutic challenge. Replacing or substituted invalid/improper acting protein offer fast and effective treatment of disease. Herein, we describe the synthesis and properties of biotinylated peptidomimetics consisting of oxoacid-modified 2,3, L-diaminopropionic acid residues with guanidine groups on its side chains. Electrophoretic analysis showed that the obtained compounds interact with FITC-labeled streptavidin or a streptavidin-ß-galactosidase hybrid in an efficient manner. Complexes formed by the abovementioned molecules are able to cross the cell membranes of cancer or healthy cells and show promising compatibility with live cells. Analysis of ß-galactosidase activity inside the cells revealed surprisingly high levels of active enzyme in complex-treated cells compared to controls. This observation was confirmed by immunochemical studies in which the presence of ß-galactosidase was detected in the membrane and vesicles of the cells.

Trehalose-based coacervates for local bioactive protein delivery to the central nervous system.

Therapeutic outcomes of local biomolecule delivery to the central nervous system (CNS) using bulk biomaterials are limited by inadequate drug loading, neuropil disruption, and severe foreign body responses. Effective CNS delivery requires addressing these issues and developing well-tolerated, highly-loaded carriers that are dispersible within local neural parenchyma. Here, we synthesized biodegradable trehalose-based polyelectrolyte oligomers using facile A2:B3:AR thiol-ene Michael addition reactions that form complex coacervates upon mixing of oppositely charged oligomers. Coacervates permit high concentration loading and controlled release of bioactive growth factors, enzymes, and antibodies, with modular formulation parameters that confer tunable release kinetics. Coacervates are cytocompatible with cultured neural cells in vitro and can be formulated to either direct intracellular protein delivery or sequester media containing proteins and remain extracellular. Coacervates serve as effective vehicles for precisely delivering biomolecules, including bioactive neurotrophins, to the mouse striatum following intraparenchymal injection. These results support the use of trehalose-based coacervates as part of therapeutic protein delivery strategies for CNS disorders.

A novel vaccine strategy against Brucellosis using Brucella abortus multi-epitope OMPs vaccine based on Lactococcus lactis live bacterial vectors.

Brucella infections typically occur in mucosal membranes, emphasizing the need for mucosal vaccinations. This study evaluated the effectiveness of orally administering Lactococcus lactis (L. lactis) for producing the Brucella abortus multi-epitope OMPs peptide. A multi-epitope plasmid was generated through a reverse vaccinology method, and mice were administered the genetically modified L. lactis orally as a vaccine. The plasmid underwent digestion, synthesizing a 39 kDa-sized protein known as OMPs by the target group. The sera of mice that were administered the pNZ8124-OMPs-L. lactis vaccine exhibited a notable presence of IgG1 antibodies specific to outer membrane proteins (OMPs), heightened levels of interferon (IFN-λ) and tumor necrosis factor alpha (TNF-α), and enhanced transcription rates of interleukin 4 (IL-4) and interleukin 10 (IL-10). The spleen sections from the pNZ8124-OMPs-L. lactis and IRIBA group had less morphological damage associated with inflammation, infiltration of lymphocytes, and lesions to the spleen. The findings present a novel approach to utilizing the food-grade, non-pathogenic L. lactis as a protein cell factory to synthesize innovative immunological candidate OMPs. This approach offers a distinctive way to evaluate experimental medicinal items' practicality, safety, affordability, and long-term sustainability.

Some Examples of Bacterial Toxins as Tools.

Pathogenic bacteria produce diverse protein toxins to disturb the host's defenses. This includes the opening of epithelial barriers to establish bacterial growth in deeper tissues of the host and to modulate immune cell functions. To achieve this, many toxins share the ability to enter mammalian cells, where they catalyze the modification of cellular proteins. The enzymatic activity is diverse and ranges from ribosyl- or glycosyl-transferase activity, the deamidation of proteins, and adenylate-cyclase activity to proteolytic cleavage. Protein toxins are highly active enzymes often with tight specificity for an intracellular protein or a protein family coupled with the intrinsic capability of entering mammalian cells. A broad understanding of their molecular mechanisms established bacterial toxins as powerful tools for cell biology. Both the enzymatic part and the pore-forming/protein transport capacity are currently used as tools engineered to study signaling pathways or to transport cargo like labeled compounds, nucleic acids, peptides, or proteins directly into the cytosol. Using several representative examples, this review is intended to provide a short overview of the state of the art in the use of bacterial toxins or parts thereof as tools.

Develop Targeted Protein Drug Carriers through a High-Throughput Screening Platform and Rational Design.

Protein-based drugs offer advantages, such as high specificity, low toxicity, and minimal side effects compared to small molecule drugs. However, delivery of proteins to target tissues or cells remains challenging due to the instability, diverse structures, charges, and molecular weights of proteins. Polymers have emerged as a leading choice for designing effective protein delivery systems, but identifying a suitable polymer for a given protein is complicated by the complexity of both proteins and polymers. To address this challenge, a fluorescence-based high-throughput screening platform called ProMatch to efficiently collect data on protein-polymer interactions, followed by in vivo and in vitro experiments with rational design is developed. Using this approach to streamline polymer selection for targeted protein delivery, candidate polymers from commercially available options are identified and a polyhexamethylene biguanide (PHMB)-based system for delivering proteins to white adipose tissue as a treatment for obesity is developed. A branched polyethylenimine (bPEI)-based system for neuron-specific protein delivery to stimulate optic nerve regeneration is also developed. The high-throughput screening methodology expedites identification of promising polymer candidates for tissue-specific protein delivery systems, thereby providing a platform to develop innovative protein-based therapeutics.

An orally administered bacterial membrane protein nanodrug ameliorates doxorubicin cardiotoxicity through alleviating impaired intestinal barrier.

The cardiotoxicity caused by Dox chemotherapy represents a significant limitation to its clinical application and is a major cause of late death in patients undergoing chemotherapy. Currently, there are no effective treatments available. Our analysis of 295 clinical samples from 132 chemotherapy patients and 163 individuals undergoing physical examination revealed a strong positive correlation between intestinal barrier injury and the development of cardiotoxicity in chemotherapy patients. We developed a novel orally available and intestinal targeting protein nanodrug by assembling membrane protein Amuc_1100 (obtained from intestinal bacteria Akkermansia muciniphila), fluorinated polyetherimide, and hyaluronic acid. The protein nanodrug demonstrated favorable stability against hydrolysis compared with free Amuc_1100. The in vivo results demonstrated that the protein nanodrug can alleviate Dox-induced cardiac toxicity by improving gut microbiota, increasing the proportion of short-chain fatty acid-producing bacteria from the Lachnospiraceae family, and further enhancing the levels of butyrate and pentanoic acids, ultimately regulating the homeostasis repair of lymphocytes in the spleen and heart. Therefore, we believe that the integrity of the intestinal barrier plays an important role in the development of chemotherapy-induced cardiotoxicity. Protective interventions targeting the intestinal barrier may hold promise as a general clinical treatment regimen for reducing Dox-induced cardiotoxicity.

Biodynamer Nano-Complexes and -Emulsions for Peptide and Protein Drug Delivery.

Background: Therapeutic proteins and peptides offer great advantages compared to traditional synthetic molecular drugs. However, stable protein loading and precise control of protein release pose significant challenges due to the extensive range of physicochemical properties inherent to proteins. The development of a comprehensive protein delivery strategy becomes imperative accounting for the diverse nature of therapeutic proteins. Methods: Biodynamers are amphiphilic proteoid dynamic polymers consisting of amino acid derivatives connected through pH-responsive dynamic covalent chemistry. Taking advantage of the amphiphilic nature of the biodynamers, PNCs and DEs were possible to be prepared and investigated to compare the delivery efficiency in drug loading, stability, and cell uptake. Results: As a result, the optimized PNCs showed 3-fold encapsulation (<90%) and 5-fold loading capacity (30%) compared to DE-NPs. PNCs enhanced the delivery efficiency into the cells but aggregated easily on the cell membrane due to the limited stability. Although DE-NPs were limited in loading capacity compared to PNCs, they exhibit superior adaptability in stability and capacity for delivering a wider range of proteins compared to PNCs. Conclusion: Our study highlights the potential of formulating both PNCs and DE-NPs using the same biodynamers, providing a comparative view on protein delivery efficacy using formulation methods.

Reversible Assembly of Proteins and Phenolic Polymers for Intracellular Protein Delivery with Serum Stability.

The design of intracellular delivery systems for protein drugs remains a challenge due to limited delivery efficacy and serum stability. Herein, we propose a reversible assembly strategy to assemble cargo proteins and phenolic polymers into stable nanoparticles for this purpose using a heterobifunctional adaptor (2-formylbenzeneboronic acid). The adaptor is easily decorated on cargo proteins via iminoboronate chemistry and further conjugates with catechol-bearing polymers to form nanoparticles via boronate diester linkages. The nanoparticles exhibit excellent serum stability in culture media but rapidly release the cargo proteins triggered by lysosomal acidity and GSH after endocytosis. In a proof-of-concept animal model, the strategy successfully transports superoxide dismutase to retina via intravitreal injection and efficiently ameliorates the oxidative stress and cellular damage in the retina induced by ischemia-reperfusion (I/R) with minimal adverse effects. The reversible assembly strategy represents a robust and efficient method to develop serum-stable systems for the intracellular delivery of biomacromolecules.

Protein-Integrated Hydrogen-Bonded Organic Frameworks: Chemistry and Biomedical Applications.

Hydrogen-bonded organic frameworks (HOFs) are porous nanomaterials that offer exceptional biocompatibility and versatility for integrating proteins for biomedical applications. This minireview concisely discusses recent advancements in the chemistry and functionality of protein-HOF interfaces. It particularly focuses on strategic methodologies, such as the careful selection of building blocks and the genetic engineering of proteins, to facilitate protein-HOF interactions. We examine the role of enzyme encapsulation within HOFs, highlighting its capability to preserve enzyme function, a crucial aspect for applications in biosensing and disease diagnosis. Moreover, we discuss the emerging utility of nanoscale HOFs for intracellular protein delivery, illustrating their applicability as nanoreactors for intracellular catalysis and neuroprotective biorthogonal catalysis within cellular compartments. We highlight the significant advancement of designing biodegradable HOFs tailored for cytosolic protein delivery, underscoring their promising application in targeted cancer therapies. Finally, we provide a perspective viewpoint on the design of biocompatible protein-HOF assemblies, underlining their promising prospects in drug delivery, disease diagnosis, and broader biomedical applications.

In vitro and in vivo characterization of human serum albumin-based PEGylated nanoparticles for BDNF and NT3 codelivery.

The utilization of neurotrophins in medicine shows significant potential for addressing neurodegenerative conditions, such as age-related macular degeneration (AMD). However, the therapeutic use of neurotrophins has been restricted due to their short half-life. Here, we aimed to synthesize PEGylated nanoparticles based on electrostatic-driven interactions between human serum albumin (HSA), a carrier for adsorption; neurotrophin-3 (NT3); and brain-derived neurotrophic factor (BDNF). Electrophoretic (ELS) and multi-angle dynamic light scattering (MADLS) revealed that the PEGylated HSA-NT3-BDNF nanoparticles ranged from 10 to 430 nm in diameter and exhibited a low polydispersity index (<0.4) and a zeta potential of -8 mV. Based on microscale thermophoresis (MST), the estimated dissociation constant (Kd) from the HSA molecule of BDNF was 1.6 µM, and the Kd of NT3 was 732 µM. The nanoparticles were nontoxic toward ARPE-19 and L-929 cells in vitro and efficiently delivered BDNF and NT3. Based on the biodistribution of neurotrophins after intravitreal injection into BALB/c mice, both nanoparticles were gradually released in the mouse vitreous body within 28 days. PEGylated HSA-NT3-BDNF nanoparticles stabilize neurotrophins and maintain this characteristic in vivo. Thus, given the simplicity of the system, the nanoparticles may enhance the treatment of a variety of neurological disorders in the future.

Development of nano-liposomal human growth hormone as a topical formulation for preventing uvb-induced skin damage.

Due to its involvement in skin maintenance and repair, topical administration of recombinant human growth hormone (rhGH) is an interesting strategy for therapeutic purposes. We have formulated and characterized a topical rhGH-loaded liposomal formulation (rhGH-Lip) and evaluated its safety, biological activity, and preventive role against UVB-induced skin damage. The rhGH-Lip had an average size and zeta potential of 63 nm and -33 mV, respectively, with 70 % encapsulation efficiency. The formulation was stable at 4 °C for at least one year. The SDS-PAGE and circular dichroism results showed no structural alterations in rhGH upon encapsulation. In vitro, studies in HaCaT, HFFF-2, and Ba/F3-rhGHR cell lines confirmed the safety and biological activity of rhGH-Lip. Franz diffusion cell study showed increased rhGH skin permeation compared to free rhGH. Animal studies in nude mice showed that liposomal rhGH prevented UVB-induced epidermal hyperplasia, angiogenesis, wrinkle formation, and collagen loss, as well as improving skin moisture. The results of this study show that rhGH-Lip is a stable, safe, and effective skin delivery system and has potential as an anti-wrinkle formulation for topical application. This study also provides a new method for the topical delivery of proteins and merits further investigation.