A Solvent-Free Thermosponge Nanoparticle Platformfor Efficient Delivery of Labile Proteins

Despite the long list of therapeutic proteins available for treating various human diseases, the vast majority of commercial protein-based drugs, such as cytokines, hormones, and monoclonal antibodies, have been limited to extracellular mechanisms of action. Many intracellular proteins with great potential as biopharmaceutical drugs have been identified; however, many of the challenges associated with intracellular protein delivery have yet to be solved. Although protein transduction and lipid nanoparticle-mediated protein delivery methods have been proposed for direct protein delivery into target cells and tissues, many obstacles remain before these methods can be successfully employed in vivo, including low purification efficiency, failure to separate from nanoparticles in recipient cells, and induction of immune responses against host immune cells. To address these limitations, we developed an opto-genetically engineered exosome system, named ‘exosomes for protein loading via optically reversible protein-protein interaction” (EXPLOR) that can deliver soluble proteins into the cytosol via controlled, reversible protein-protein interactions (PPI). Among nanoparticles, cell-derived exosomes have recently been highlighted as new therapeutic strategies for the in vivo delivery of nucleotides and chemical drugs. Exosomes are natural cell-derived extracellular vesicles that originate from internal endocytic compartments and multi-vesicular bodies and participate in intercellular communication. Recent studies have sought to use exosomes as a new method for the in vivo delivery of siRNA or miRNA to specific target tissues by systemic injection. These methods were based on the passive loading of siRNAs or miRNAs into isolated exosomes by electrophoresis, a method poorly suited for the intracellular delivery of cellular proteins. By integrating a reversible PPI module controlled by blue light with the endogenous process of exosome biogenesis, we were able to successfully load cargo proteins into newly generated exosomes. Treatment with protein-loaded EXPLORs was shown to significantly increase intracellular levels of cargo proteins and their function in recipient cells in both a time- and dose-dependent manner. In the present study, we have demonstrated the intracellular delivery of mCherry, Cre enzyme, Bax, and Super repressor IκB proteins as functional proteins in the target cells and in vivo. These results clearly indicate the potential of EXPLORs as a mechanism for the efficient intracellular transfer of protein-based drugs into recipient cells and tissues both in vitro and in vivo. Citation Format: Nambin Yim, Seung-Wook Ryu, Kyungsun Choi, Kwang Ryeol Lee, Seunghee Lee, Hojun Choi, Jiho Park, Daesoo Kim, Wondo Heo, Chulhee Choi. Efficient and rapid cellular delivery of bioactive proteins using EXPLOR: exosomes engineered for protein loading via optically reversible protein-protein interaction. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2167.

Efficient intracellular delivery of native proteins facilitated by preorganized guanidiniums on pillar[5]arene skeleton

Peptide and protein drugs have found an important position in therapeutics. Recent advances in pharmaceutical biotechnology have led to an increase in the number of protein products in the market. As these therapeutic proteins and peptides are made available, it will be essential to formulate these drugs into safe and effective delivery systems. The twenty different naturally occurring amino acids join with each other by peptide bonds and build polymers referred to peptides and proteins. Although the distinction between peptides and proteins is arbitrary, a peptide contains less than 20 amino acids, having a molecular weight less than 5000, while a protein possesses 50 or more amino acids and its molecular weight lies above this value. The most important challenge to the formulation of peptides and proteins into effective dosage forms is to ensure their stability over their shelf lives. Physical instability (including denaturation, aggregation, precipitation and adsorption onto surfaces) and chemical instability (including oxidation, hydrolysis, deamidation, beta-elimination, racemization and disulfide exchange) may occur for a given peptide or protein, due to the presence of multiple susceptible sites. It should be noted that in most cases, more than one pathway of physical and/or chemical instability may be responsible for the degradation of peptides and proteins. Therefore, compared to the formulation of traditional dosage forms, formulation of peptide and protein drugs is very difficult and, regardless of the route of administration, product development should start with preformulation studies including physicochemical characterization, solubility determination, stability determination under various conditions, isoelectric point determination, optical pH determination and characterization of impurities. Also, choice of buffer system, pH of the vehicle, selection of an appropriate solvent system and preservation of the formulation, as well as selection of appropriate pharmaceutical excipients, are among the factors that should be considered in the formulation development of peptides and proteins, in order to prevent or minimize the various physical and chemical degradation pathways. Although most protein pharmaceuticals are usually formulated as a solution or suspension and delivered by invasive routes such as subcutaneous injections, major efforts in both academic and industrial laboratories have been directed toward developing effective oral formulations and increasing the oral absorption of intact protein through the use of formulations that protect the macromolecule and/or enhance it’s uptake into the intestinal mucosa. However, in spite of these major attempts, relatively little progress has been made. For the efficient delivery of peptides and proteins by non-parenteral route, in particular via the gastrointestinal tract, novel concepts are needed to overcome significant enzymatic and diffusion barriers. In general, the difficulties associated with developing effective oral peptides and proteins formulations are normally ascribed to a) poor intrinsic permeability across biological membranes due to the hydrophobic nature and large molecular size, b) susceptibility to enzymatic attack, c) rapid post-absorptive clearance, and d) chemical instability. It is quite appreciated that not all peptides and proteins with therapeutic applications will need to be absorbed from the gastrointestinal tract. Those substances used exclusively in life threatening situations for a short period of time and those with rapid renal clearance or metabolism can be administered by injection, while for substances that are required prophylactically or for the control of nonvital functions, gastrointestinal absorption is most desirable. On the other hand, it is also evident that macromolecules such as proteins are absorbed from the GI tract, but only in small quantities. To overcome this problem, a variety of permeation enhancers, including salicylates, mixed bile salts fatty acid micelles, chelators, fatty acids, surfactants and medium chain glycerides have received considerable attention in an attempt to increase the absorption of peptides. These enhancers are capable of modifying the basic barrier properties of the intestinal epithelial cell membrane. However, it has been shown that manipulation of formulation variables in order to enhance the absorption of these compounds has varying degrees of success so that the bioavailability of these products is still fairly low and unpredictable. Two approaches for providing protection against proteolytic attacks are a) chemical modification of small peptides and b) protecting peptides or proteins in physical environment of formulation itself, prior to absorption, using microemulsions, nanoparticles and bioadhesive particles. Due to the complex nature of peptides and proteins, self aggregation is a major concern in formulation efforts. It has been shown that the use of surfactants could maximize monomer concentration during peptide and protein release and minimize the size of complex for permeation through epithelial cell layer. Peptides and proteins play a key role in physiological activities (e.g. reproduction, growth, etc.) and have been used for the treatment of various pathologic conditions such as diabetes mellitus, endocrine disorders, autoimmune disorders and specific metabolic abnormalities. Recent developments in the field of biotechnology, recombinant DNA technology and analytical methods for peptides and proteins are resulting in a greater availability of peptides and proteins for therapeutic use. It seems that the enzymatic degradation and poor administration of macromolecules from the gastrointestinal tract have limited the administration of peptides and proteins to the parenteral route. However, in the past few years, new advances have been achieved in the delivery of peptides and proteins via mucosal (nasal, pulmonary, rectal and ocular), transdermal and topical routes, as well as oral administration. Regardless of the route of administration, the aims of a formulator are to maintain the stability of peptides and proteins prior to their absorption and localization at or near the target site, decrease their antigenicity properties, prolong their half-life and increase their absorption through biological membranes. The scientific community has reached a new stage in the understanding of the properties of peptides and proteins and in the manufacturing of these therapeutic agents. In the past, administration of peptides and proteins was believed to be impossible, while nowadays it is expected that the obstacles for effective delivery of therapeutic peptides and proteins will be overcome and more products would be made available to the patients.

Recent advances in genetic engineering and pharmaceutical biotechnology have made possible to combat life-threatening diseases with efficient delivery of therapeutic proteins. These advancements have increased the significance of therapeutic proteins in pharmaceutical market, but their therapeutic delivery to the targeted site is still a major obstacle to achieve desired therapeutic outcomes. In most cases, majority of the therapeutic proteins are usually administered via oral routes which encounter many problems notably enzymatic degradation, poor solubility and nonlinear pharmacokinetics. Besides this route, many other routes like mucosal, intra-nasal, intra-vaginal, pulmonary and transdermal have also been used for the delivery of therapeutic proteins. In order to keep these therapeutic proteins safe from enzymatic degradation and improve their therapeutic efficacy, several strategies have been designed and investigated various therapeutic delivery routes for efficient delivery of therapeutic proteins to the targeted site with minimal side effects. In this article, we have comprehensively summarized the recent advances and developments that have been adopted for delivery systems of these therapeutic proteins via invasive and/or non-invasive routes.

In this work, the importance of the size and concentration of binding sites in mesoporous nanostructures on their performance for protein delivery is reported. Various types of carboxyl-functionalized mesoporous silica (MS) nanoparticles, i.e., SA(x)-MS, PA1(x)-MS and PA2(x)-MS, where x = 5, 10, 15, 20 and 30 mg, were synthesized by using succinic anhydride (SA) and polyacrylic acid with two molecular weights (i.e., 1800 (PA1) and 100,000 (PA2)), respectively. The particles were thoroughly characterized by TGA, FTIR, STEM and zeta potential measurement techniques and then, their biomedical performance was investigated by using a model therapeutic protein, lysozyme. The carboxyl functional molecules concentration (CCOOH) on the particles was increased as a function of the functional molecules type and quantity in the synthesis reaction environment. The maximum CCOOH values were recorded for SA(10)-MS, PA1(15)-MS and PA2(15)-MS, being 827, 1662 and 2137 µmol.g−1, respectively. However, SA(10)-MS, PA1(15)-MS and PA2(10)-MS particles led to the highest lysozyme loading yield values of 63.1%, 90.0% and 71.5% as well as the loading capacities of 631, 900 and 715 mg.g−1, respectively. The controlled lysozyme release rate and some protein conformational changes favored the in vitro antibacterial activity of lysozyme molecules carried by the particles, which followed the order of PA1(15)-MS > PA2(10)-MS > SA(10)-MS.

Catechol-Based Polymers with High Efficacy in Cytosolic Protein Delivery

Affinity-controlled protein encapsulation into sub-30nm telodendrimer nanocarriers by multivalent and synergistic interactions.

Efficient intracellular and in vivo delivery of toxin proteins by a ROS-responsive polymer for cancer therapy.

Efficient intracellular delivery of p53 protein by engineered protein crystals restores tumor suppressing function in vivo

The delivery of protein and peptide therapeutics through ocular route requires considerable knowledge and understanding about eye’s anatomy and physiology. Although this delivery route has high potency and specificity, it exhibits difficulty in absorption through barriers resulting in lower bioavailability as well as crucial stability issues. Due to the complications associated with the most common injectable route for the peptide and protein delivery, there is a surge for the noninvasive route such as ocular which include intravitreal and periocular route. Newer techniques for delivery of these macromolecules involve targeting transporters or receptors to enhance specificity, while approaches such as nanocarriers, prodrug, mucoadhesion, and permeation enhancers have been employed to attain enhanced bioavailability. This chapter addresses pros and cons of ocular delivery of peptides and proteins, significant features of their chemistry, potential and challenges associated with their local and systemic delivery, as well as different ways to attain better protein bioavailability and stability.

Biofunctional proteins such as active enzymes and therapeutic proteins show tremendous promise in disease treatment. However, intracellular delivery of proteins is facing substantial challenges owing to their vulnerability to degradation and denaturation and the presence of various biological barriers such as their low membrane transport efficiency. Herein, we report a magnetically driven and redox-responsive nanotransporter (MRNT) for highly efficient intracellular delivery of biofunctional proteins. The MRNT has remarkably high cargo capacity, compared with that without nanoscale cargo compartments. We have demonstrated the directional and dynamic motion of the MRNT using both nanoparticle tracking analysis and magnetic driving evaluation. Moreover, the active MRNT can translocate into the cytosol and sense the reducing cytosolic environment to discharge protein cargoes autonomously. The internalization mechanism of the MRNT has been studied using endocytosis inhibitors. Under the magnetic drive, the MRNT can promote a protein transduction efficiency of over 95%, and the intracellular protein delivery by the active MRNT shows significantly higher (∼4 times) enzymatic activity and therapeutic efficiency than those achieved by the static ones. Our proof-of-concept study provides a valuable tool for intracellular protein transduction and contributes to biotechnology and protein therapeutics.

Efficient and safe intracellular delivery of proteins is highly desired in the development of protein therapeutics. Current methods of protein delivery commonly suffer from low loading efficiency, low stability in serum, and lack of versatility for different proteins. Here, we developed a platform for efficient protein delivery using mesoporous silica nanoparticles (MSN) with lipid fusion. By different surface modifications on MSN, the positively charged MSN (MSN+) and the negatively charged MSN (MSN-), were generated for loading different proteins. The cargo proteins, based on the surface charges, can be selectively loaded in very high efficiency. The protein-loaded MSNs were fused with liposomes to form a protocell-like delivery system (MSN-LP) in order to prevent burst release of proteins. The lipid fusion significantly increases the stability of the nanosystem in physiological conditions, and the MSN-LP protocell can efficiently deliver proteins into cells. The cargo proteins can be released in cells in a sustained manner. Fifteen different proteins, including two protein complexes, were tested using this delivery system. Further analyses indicate that the proteins can maintain their functions after delivery into cells. Fluorescent proteins, GFP, and KillerRed show fluorescence in cells, indicating the correct folding of proteins during encapsulation and delivery. Protein activity analysis shows that KillerRed protein can generate ROS in cells, while SOD can eliminate ROS in cells. Hence, the proteins delivered by this system remain their structure and function in cells. This work provides a versatile strategy for charge-selective delivery of proteins with high loading efficiency and high stability.

Therapeutic proteins are playing increasingly important roles in treating numerous types of diseases. However, oral administration of proteins, especially large ones (e.g., antibodies), remains a great challenge due to their difficulties in penetrating intestinal barriers. Herein, fluorocarbon-modified chitosan (FCS) is developed for efficient oral delivery of different therapeutic proteins, in particular large ones such as immune checkpoint blockade antibodies. In our design, therapeutic proteins are mixed with FCS to form nanoparticles, lyophilized with appropriate excipients, and then filled into enteric capsules for oral administration. It has been found that FCS could promote transmucosal delivery of its cargo protein via inducing transitory rearrangement of tight junction associated proteins between intestinal epithelial cells and subsequently release free proteins into blood circulation. It is shown that at a 5-fold dose oral delivery of anti-programmed cell death protein-1 (αPD1) or its combination with anti-cytotoxic T-lymphocyte antigen 4 (αCTLA4) using this method could achieve comparable antitumor therapeutic responses to that achieved by intravenous injection of corresponding free antibodies in various types of tumor models and, more excitingly, result in significantly reduced immune-related adverse events. Our work successfully demonstrates the enhanced oral delivery of antibody drugs to achieve systemic therapeutic responses and may revolutionize the future clinical usage of protein therapeutics.

Efficient cytosolic protein delivery is necessary to fully realize the potential of protein therapeutics. Current methods of protein delivery often suffer from low serum tolerance and limited in vivo efficacy. Here, we report the synthesis and validation of a previously unreported class of carboxylated branched poly(β-amino ester)s that can self-assemble into nanoparticles for efficient intracellular delivery of a variety of different proteins. In vitro, nanoparticles enabled rapid cellular uptake, efficient endosomal escape, and functional cytosolic protein release into cells in media containing 10% serum. Moreover, nanoparticles encapsulating CRISPR-Cas9 ribonucleoproteins (RNPs) induced robust levels of gene knock-in (4%) and gene knockout (>75%) in several cell types. A single intracranial administration of nanoparticles delivering a low RNP dose (3.5 pmol) induced robust gene editing in mice bearing engineered orthotopic murine glioma tumors. This self-assembled polymeric nanocarrier system enables a versatile protein delivery and gene editing platform for biological research and therapeutic applications.

Therapeutic proteins and peptides have become notable in the drug delivery arena for their compatibility with the human body as well as their high potency. However, their biocompatibility and high potency does not negate the existence of challenges resulting from physicochemical properties of proteins and peptides, including large size, short half-life, capability to provoke immune responses and susceptibility to degradation. Various delivery routes and delivery systems have been utilized to improve bioavailability, patient acceptability and reduce biodegradation. The ocular route remains of great interest, particularly for responsive delivery of macromolecules due to the anatomy and physiology of the eye that makes it a sensitive and complex environment. Research in this field is slowly gaining attention as this could be the breakthrough in ocular drug delivery of macromolecules. This work reviews stimuli-responsive polymeric delivery systems, their use in the delivery of therapeutic proteins and peptides as well as examples of proteins and peptides used in the treatment of ocular disorders. Stimuli reviewed include pH, temperature, enzymes, light, ultrasound and magnetic field. In addition, it discusses the current progress in responsive ocular drug delivery. Furthermore, it explores future prospects in the use of stimuli-responsive polymers for ocular delivery of proteins and peptides. Stimuli-responsive polymers offer great potential in improving the delivery of ocular therapeutics, therefore there is a need to consider them in order to guarantee a local, sustained and ideal delivery of ocular proteins and peptides, evading tissue invasion and systemic side-effects.