Alternative Excipients for Protein Stabilization in Protein Therapeutics: Overcoming the Limitations of Polysorbates.

Given their safety and efficiency in protecting protein integrity, polysorbates (PSs) have been the most widely used excipients for the stabilization of protein therapeutics for years. In recent decades, however, there have been numerous reports about visible or sub-visible particles in PS-containing biotherapeutic products, which is a major quality concern for parenteral drugs. Alternative excipients that are safe for parenteral administration, efficient in protecting different protein drugs against various stress conditions, effective in protein stabilization in high-concentrated liquid formulations, stable under the storage conditions for the duration of the product's shelf-life, and compatible with other formulation components and the primary packaging are highly sought after. The aim of this paper is to review potential alternative excipients from different families, including surfactants, carbohydrate- and amino acid-based excipients, synthetic amphiphilic polymers, and ionic liquids that enable protein stabilization. For each category, important characteristics such as the ability to stabilize proteins against thermal and mechanical stresses, current knowledge related to the safety profile for parenteral administration, potential interactions with other formulation components, and primary packaging are debated. Based on the provided information and the detailed discussion thereof, this paper may pave the way for the identification or development of efficient excipients for biotherapeutic protein stabilization.

Adalimumab Decorated Nanoparticles Enhance Antibody Stability and Therapeutic Outcome in Epithelial Colitis Targeting.

Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract with increasing incidence worldwide. Although a deeper understanding of the underlying mechanisms of IBD has led to new therapeutic approaches, treatment options are still limited. Severe adverse events in conventional drug therapy and poor drug targeting are the main cause of early therapy failure. Nanoparticle-based targeting approaches can selectively deliver drugs to the site of inflammation and reduce the risk of side effects by decreasing systemic availability. Here, we developed a nanoparticulate platform for the delivery of the anti-TNF-α antibody adalimumab (ADA) by covalent crosslinking to the particle surface. ADA binding to nanoparticles improved the stability of ADA against proteolytic degradation in vitro and led to a significantly better therapeutic outcome in a murine colitis model. Moreover, immobilization of ADA reduced systemic exposure, which can lead to enhanced therapeutic safety. Thus, nanoparticle protein decoration constitutes a platform through which epithelial delivery of any biological of interest to the inflamed gut and hence a local treatment can be achieved.

Drug delivery to the inflamed intestinal mucosa - targeting technologies and human cell culture models for better therapies of IBD.

Current treatment strategies for inflammatory bowel disease (IBD) seek to alleviate the undesirable symptoms of the disorder. Despite the higher specificity of newer generation therapeutics, e.g. monoclonal antibodies, adverse effects still arise from their interference with non-specific systemic immune cascades. To circumvent such undesirable effects, both conventional and newer therapeutic options can benefit from various targeting strategies. Of course, both the development and the assessment of the efficiency of such targeted delivery systems necessitate the use of suitable in vivo and in vitro models representing relevant pathophysiological manifestations of the disorder. Accordingly, the current review seeks to provide a comprehensive discussion of the available preclinical models with emphasis on human in vitro models of IBD, along with their potentials and limitations. This is followed by an elaboration on the advancements in the field of biology- and nanotechnology-based targeted drug delivery systems and the potential rooms for improvement to facilitate their clinical translation.

Polymeric matrix hydrophobicity governs saponin packing-density on nanoparticle surface and the subsequent biological interactions.

This study investigated the loading behavior of Quillaja saponin as a model surface-active cargo on (NP) nanoparticles prepared with various hydrophobic polymers and using different organic solvents through emulsification/solvent evaporation, and the impact of NP surface hydrophobicity upon the cytotoxic and hemolytic properties of the loaded entity. A superficial monolayered arrangement of saponins on NP was established (R2 > 0.9) for all NP, as the saponin loading values complied with the Langmuir adsorption isotherm over the entire concentration range. Next, based on the measurement of interfacial tension between formulation phases, and the subsequent use of Gibb's adsorption isotherm, the packing density (Гexc) and loading of saponins on various nanospheres could be predicted with good correlation with the actual values (R2 > 0.95). The results demonstrated that the hydrophobicity of the polymeric matrix was the major determinant of saponin packing density on the nanospheres. Finally, the impact of NP surface properties upon saponin biological interactions was investigated, where a linear correlation was found between the NP surface hydrophobicity and their hemolytic properties (R2 â‰… 0.79), and cytotoxicity against two cancer cell lines (R2 > 0.76). The surface hydrophobicity of the polymeric NP seemingly governed the NP-cell membrane binding, which in turn determined the amount of membrane-bound saponins per unit NP surface area. As the saponins exert their cytotoxicity mainly through strong permeabilization of the cell membrane, a higher amount of NP-membrane association governed by a more hydrophobic matrix can lead to higher levels of cytotoxicity. These findings highlight the importance of a detailed characterization of NP surface properties, particularly in case of surface-active cargos, for these dictate the side effects and biological interactions of the delivery system.

Nanotechnology as a Platform for the Development of Injectable Parenteral Formulations: A Comprehensive Review of the Know-Hows and State of the Art.

Within recent decades, the development of nanotechnology has made a significant contribution to the progress of various fields of study, including the domains of medical and pharmaceutical sciences. A substantially transformed arena within the context of the latter is the development and production of various injectable parenteral formulations. Indeed, recent decades have witnessed a rapid growth of the marketed and pipeline nanotechnology-based injectable products, which is a testimony to the remarkability of the aforementioned contribution. Adjunct to the ability of nanomaterials to deliver the incorporated payloads to many different targets of interest, nanotechnology has substantially assisted to the development of many further facets of the art. Such contributions include the enhancement of the drug solubility, development of long-acting locally and systemically injectable formulations, tuning the onset of the drug's release through the endowment of sensitivity to various internal or external stimuli, as well as adjuvancy and immune activation, which is a desirable component for injectable vaccines and immunotherapeutic formulations. The current work seeks to provide a comprehensive review of all the abovementioned contributions, along with the most recent advances made within each domain. Furthermore, recent developments within the domains of passive and active targeting will be briefly debated.

Nanosphere-shaped ammonio methacrylate copolymers: converting a pharmaceutical inactive ingredient to efficient therapeutics for experimental colitis.

Inflammatory bowel disease (IBD) refers to progressive inflammatory disorders that impair the gastrointestinal tract's structure and function. Given their selective accumulation in inflamed tissues, nanoparticles are promising drug delivery systems for IBD treatment. The hypothesis here was that drug-free nanoscaled cationic ammonio methacrylate copolymers (AMCNP) may have a beneficial therapeutic effect in murine TNBS-induced colitis. Type A and B AMCNP (RLNP and RSNP, respectively) were prepared and characterized in vitro, and were rectally administered in two concentrations (5 and 25 mg ml-1) for the treatment of two grades of murine experimental colitis. The impact of the nanoparticles upon the inflammatory markers, circulating LPS, intestinal permeability and colonic leukocyte populations was examined. Both RLNP and RSNP led to a significant mitigation of mild to moderate experimental colitis, as evident from the substantial reduction of myeloperoxidase (MPO) and alkaline phosphatase (AP) activities (more than two-fold, P < 0.05) and various pro-inflammatory cytokine concentrations (TNF-α, IL-1ß, IL-6, IL-12). The best therapeutic efficiency was observed when the particles were used at 5 mg ml-1, while the more cationic RLNP performed superior. When used against a severe grade of colitis, RLNP (5 mg ml-1) resulted in a significant decrease of tissue MPO and TNF-α. It was found that treatment with AMCNP resulted in significant intestinal immune cell depletion, intestinal barrier function improvement, and 1.5-2.5 times reduction of the systemic endotoxin concentration. These findings highlighted the fact that nanoscaling endows the cationic amphiphilic AMCs unique therapeutic properties, which help mitigate murine experimental colitis in the absence of any drug load. The results also provided a glimpse of possible underlying mechanisms through which nanoscaled AMCs might have exerted their therapeutic effect within this context.

Collagenase loaded chitosan nanoparticles for digestion of the collagenous scar in liver fibrosis: The effect of chitosan intrinsic collagen binding on the success of targeting.

A variety of hepatic insults result in the accumulation of collagen-rich new extracellular matrix in the liver, ultimately culminating in liver fibrosis and cirrhosis. For such reasons, approaches looking into digestion of the collagen-rich extracellular matrix present an interesting therapeutic approach for cases of chronic liver disease, where the fibrotic scar is well established. Portal collagenase administration has recently led to the successful reversion of cirrhosis in an experimental rabbit model. Notwithstanding, the question of how such a sensitive therapeutic macromolecule could be administered in a less invasive manner, and in a way that preserves its functionality and avoids digestion of other non-hepatic vital collagen presents itself. Chitosan is a biodegradable polymer that has been reported to interact and bind to collagen. Chitosan nanoparticles (CS NPs) have also been reported to encapsulate therapeutic proteins, maintaining their functional form and protecting them from in-vivo degradation. For such reasons, CS NPs were loaded with collagenase and evaluated in-vitro and in-vivo for their ability to target and digest collagen. CS NPs were able to encapsulate collagenase (≈ 60% encapsulation efficiency) and release its content in active form. To determine whether chitosan's collagen interaction would enable NP collagen binding or whether the modification with collagen binding peptides (CBPs) is necessary, CS NPs were modified with the CBP; CCQDSETRTFY. Since the density of targeting ligand and the length of tether play a significant role in the success of active targeting, the surface of NPs was modified with different densities of the CBP either directly or using a polyethylene glycol (PEG) spacer. PEGylated NPs showed higher levels of CBP tagging; high, intermediate and low density of CBPs corresponded to 585.8 ± 33, 252.9 ± 25.3 and 56.5 ± 8.8 µg/mL for PEGylated NPs and 425.56 ± 12.67, 107.91 ± 10.3 and 49.86 ± 3.2 µg/mL for unPEGylated NPs, respectively. In-vitro collagen binding experiments showed that unmodified CS NPs were able to bind collagen and that modification with CBPs either directly or via PEG did not enhance collagen binding. In-vivo experiments demonstrated that unmodified CS NPs were able to reverse fibrosis with a survival rate of 100% at the end of the study, indicating the ability of CS NPs to deliver functional collagenase to the fibrotic liver and making the use of CBPs unnecessary.

Liposomal delivery of functional transmembrane ion channels into the cell membranes of target cells; a potential approach for the treatment of channelopathies.

Defects in transmembrane ion channels underlie many disorders, commonly known as channelopathies. Current therapies are mostly symptomatic and do not treat the underlying cause. Here, we demonstrate the delivery of functional ion channels in protein form into the membrane of target cells using fusogenic proteoliposomes. The glycine receptor (GlyR) was adopted as a model channel. HEK293 cells were transfected with GlyR and GlyR-rich cell membrane fragments (CMF) were incorporated into fusogenic liposomes. Proteoliposomes were generated using 1,2-dioleoylphosphoethanolamine (DOPE) as the fusogenic lipid, lecithin, 1,2-distearoylphosphoethanolamine (DSPE), and cholesterol (Chol). Three formulations were prepared Non-fuse (2.5:0.5 Lecithin: Chol), Fuse1 (1.25:0.25:0.25:0.25) and Fuse2 (1.25:0.5:0.5:0.25 Lecithin: DOPE: DSPE: Chol). Proteoliposomes were assessed for their ability to (1) incorporate GlyR rich CMF (2) fuse with L929 fibroblast cell membrane and (3) deliver functional GlyR to these cells. All formulations were capable of integrating CMF, with Fuse2 showing highest CMF incorporation (1.2 and 1.4 folds relative to Non-fuse and Fuse1 respectively). All liposomes showed ability to fuse with the fibroblast cell membrane, with Fuse2 showing highest fusion. Patch-clamp analysis demonstrated successful delivery of functional GlyR into the fibroblast cell membrane. Thus, proof of principle was established for the use of liposomes to deliver functional ion channels to living cells.

Modulation of Nanostructure-Based Lipopolysaccharide Active Immunotherapy in Cancer: Size and Composition Determine Short- and Long-Term Tolerability.

Despite holding promise for cancer immunotherapy, the strong pro-inflammatory properties of lipopolysaccharide (LPS) also account for severe localized and systemic side effects, restricting its administrable dosage and the possibility of chronic dosing. Herein, we exploited the surface-active properties of LPS molecules to develop pathogen-mimicking LPS-decorated nanostructures with different compositions (lipid nanoemulsion vs polymeric nanospheres) and sizes (volumetric mean diameters of 100 nm vs 700 nm). The formulations were tested in cell culture for their immunostimulatory properties and in vivo against a murine subcutaneous colorectal cancer model. While all nanostructures resulted in similar levels of apoptotic cell death in tumor cells cultured with splenocytes, both the size and the composition of the nanostructures were found to govern the short- and long-term tolerability of LPS-based immunotherapy in vivo. The toxicity-related end point of the animal trials was decided upon in the case of a body condition score (BCS) of 1 and poor hair coat, or more than 15% loss of the original body weight, while in the absence of long-term intolerability, the experiments were terminated in the case of full remission or once the tumor surpassed a volume of 1000 mm3. Size was an important determinant of short-term tolerability, with larger particles being associated with higher incidence and extent of localized necrosis (3-6% necrotic surface area). Nanostructure composition, on the other hand, predominantly governed the long-term systemic tolerability. Within this context, the higher affinity of LPS molecules to the triglyceride core of the nanoemulsion compared to the polymeric matrix significantly improved the tolerability of the former over time. In fact, the mean survival estimate of the animals treated with small LPS nanoemulsion (LPS-NE (small)) was at least 42 days longer than that of the LPS and the LPS-decorated polymeric nanoparticle (LPS-NP) groups. Unlike other treatment groups, the experiments on 80% of the animals in LPS-NE (small) were terminated due to complete remission or tumor volume >1000 mm3. While a better understanding of these findings requires a larger scale, mechanistic-oriented trial on larger animal models, they indicate the role of nanostructures as beyond the carriers of the incorporated immunotherapeutic cargos. This highlights the importance of a wise selection of nanoparticle composition and a purposeful tuning of their physicochemical properties to enhance the safety profile and improve the eventual immunotherapeutic outcome.

Sub-cytotoxic doses of pharmaceutical silica nanoparticles show significant impact on the proteome of HepG2 cells.

The ever-growing application of nanoparticles (NPs) in medical and pharmaceutical domains has brought issues related to their toxicity into focus. However, a profound analysis of non-acute, sub-lethal effects of engineered pharmaceutical NPs is often disregarded during such toxicological investigations. Here, two selected NPs were investigated in cultured HepG2 cells in terms of their intracellular localization and the associated impact on pharmacokinetically relevant CYP3A4 isoform, as well as the induced changes observed in the proteome of such cells. Using SILAC (Stable Isotope Labeling by Amino acids in Cell culture)-based mass spectrometry facilitated quantitative proteomics, significant proteomic changes in NP-treated hepatocytes were detected, which were subsequently analyzed via bioinformatic tools. Both, silica NPs (SiO2 NP) and cargo-free PEGylated stealth liposomes resulted in the induction of CYP3A4-activity up to 150% in a dose-dependent manner, with different time-dependent response-patterns as a function of NP-type after a single treatment. Proteomic analysis revealed that the observed metabolic alterations are only one aspect of the cellular response to NP-exposure. SiO2NPs (free in cytoplasm) caused extensive changes in the proteome, whereas liposomes (compartmentalized) seemed unproblematic as they accounted for minimal changes in the protein profile. Based on the obtained results, proteomic analyses were revealed to be highly important for the toxicological assessment of NPs. Although sub-toxic concentrations of many NPs are considered as uncritical based on standard toxicological assays, proteomic analysis indicated that drug-free NPs could cause fundamental cellular modifications, which were attributed to different causal networks and regulatory pathways.

Challenges of using lipopolysaccharides for cancer immunotherapy and potential delivery-based solutions thereto.

Despite being one of the earliest Toll-like receptor (TLR)-based cancer immunotherapeutics discovered and investigated, the full extent of lipopolysaccharide (LPS) potentials within this arena remains hitherto unexploited. In this review, we will debate the challenges that have complicated the improvement of LPS-based immunotherapeutic approaches in cancer therapy. Based on their nature, those will be discussed with a focus on side effect-related, tolerance-related and in vivo model-related challenges. We will then explore how drug delivery strategies can be integrated within this domain to address such challenges in order to improve the therapeutic outcome, and will present a summary of the studies that have been dedicated thereto. This paper may inspire further developments based on reconciling the advantages of drug delivery and LPS-based cancer immunotherapy.

TLR4-Based Immunotherapeutics in Cancer: A Review of the Achievements and Shortcomings.

Toll-like Receptor 4 (TLR4) agonists have had a long journey in the field of cancer immunotherapy. Nevertheless, despite the remarkable number of the TLR4 ligands that have gone through various preclinical and clinical stages, only two (Bacillus Calmette-Guérin (BCG) and monophosphoryl lipid A (MPLA)) have hitherto obtained the FDA approval for clinical application in cancer treatment. This paper provides a comprehensive review of the TLR4 agonists' journey as cancer active immunotherapeutics. Following a brief discussion of the rationale behind the use of TLR ligands in cancer immunotherapy, we will initially focus on the forerunner of the TLR4 agonists, bacterial lipopolysaccharide (LPS). Within this context, the potentials and shortcomings of immunotherapy with this agent will be addressed, the strategies that have been devised to enhance the associated therapeutic outcome will be discussed, and the consequent achievements and shortcomings will be summarized. Subsequently, further and perhaps less well-known, molecular, bacterial, and viral TLR4 agonists with potential for cancer immunotherapy will be introduced, and if present, the outcome of the preclinical and clinical investigations of these agents will be reviewed. Finally, a look will be cast upon the promising souvenirs of the relatively new arena of nanotechnology, where TLR4 activating nanoparticulate systems will be proposed as potential candidates for the future development of this field.

A nanoparticle-based approach to improve the outcome of cancer active immunotherapy with lipopolysaccharides.

This study sought to develop a simple nanoparticle-based approach to enhance the efficiency and tolerability of lipopolysaccharide (LPS), a potent ligand of Toll-like Receptor 4 (TLR4), for immunotherapy in cancer. Despite holding promise within this context, the strong pro-inflammatory properties of LPS also account for its low tolerability given localized and systemic side effects, which restrict the administrable dosage. Herein, we investigated the effect of LPS decoration as a surface-active molecule on a polymeric matrix upon its efficiency and tolerability. The LPS-decorated nanoparticles (LPS-NP) were about 150 nm in size, with slightly negative zeta potential (about -15 mV) and acceptable LPS incorporation (about 70%). In vitro, the particles accounted for a higher induction of apoptosis in tumor cells cultured with murine splenocytes compared to LPS solution. When used for the treatment of a murine syngeneic colorectal tumor model, higher intratumoral deposition of the particle-bound LPS was observed. Furthermore, unlike LPS solution, which accounted for localized necrosis at high concentrations, treatment of tumor-bearing animals with equivalent doses of LPS-NP was well tolerated. We propose that the observed localized necrosis can be Shwartzman phenomenon, which, due to modulated 24-h post-injection systemic TNF-α and LPS concentrations, have been avoided in case of LPS-NP. This has in turn enhanced the therapeutic efficiency and enabled complete tumor regression at concentrations at which LPS solution was intolerable. The findings indicate that nanoparticles can serve as beyond carriers for the delivery of superficially decorated LPS molecules, but impact their overall efficiency and tolerability in cancer therapy.

MDR in cancer: Addressing the underlying cellular alterations with the use of nanocarriers.

Multidrug resistance (MDR) is associated with a wide range of pathological changes at different cellular and intracellular levels. Nanoparticles (NPs) have been extensively exploited as the carriers of MDR reversing payloads to resistant tumor cells. However, when properly formulated in terms of chemical composition and physicochemical properties, NPs can serve as beyond delivery systems and help overcome MDR even without carrying a load of chemosensitizers or MDR reversing molecular cargos. Whether serving as drug carriers or beyond, a wise design of the nanoparticulate systems to overcome the cellular and intracellular alterations underlying the resistance is imperative. Within the current review, we will initially discuss the cellular changes occurring in resistant cells and how such changes lead to chemotherapy failure and cancer cell survival. We will then focus on different mechanisms through which nanosystems with appropriate chemical composition and physicochemical properties can serve as MDR reversing units at different cellular and intracellular levels according to the changes that underlie the resistance. Finally, we will conclude by discussing logical grounds for a wise and rational design of MDR reversing nanoparticulate systems to improve the cancer therapeutic approaches.