Patent Application
20230000999
The disclosed subject matter relates to delivery of nucleic acids (DNA, plasmids, oligos, small interfering RNAs [siRNA], small hairpin RNA [shRNA], microRNAs [miRNA], Clustered Regularly Interspaced Short Palindromic Repeats [CRISPR]) using a peptide-based delivery system to living cells.
Nucleic acid delivery is an approach to suppress (or enhance) the expression of a protein temporarily or permanently. The suppression of protein expression (also known as silencing) is performed by delivering double-stranded siRNAs, shRNA, miRNA, or CRISPR/Cas9. RNA interference (RNAi) is an approach to suppress (or stop) the expression of a protein temporarily or permanently. The double stranded siRNA contains a passenger and a guide strand. The guide strand is incorporated into the RNA-induced silencing complex (RISC), while the passenger strand is degraded. The guide strand acts as a complementary sequence to the messenger RNA, and therefore, binds to the targeted mRNA, which triggers the Argonaute 2 (an essential catalytic protein in RISC) to cleavage the mRNA to small pieces, which will be degraded rapidly by RNases. This process is known as the post-transcriptional gene silencing (Akinc et al., Targeted delivery of RNAi therapeutics with endogenous and exogenous ligand-based mechanisms. Mol Ther, 2010, 18(7):1357-64), This complex intervenes in the degradation process of the siRNA sense strand and utilizes the preserved anti-sense strand to identify the complement sequences of mRNA.
Clinical evaluation of nucleic acids has been challenging due to their limited cellular uptake (because of large size and negatively charged phosphate groups in their structure), off-target effects, and rapid enzymatic degradation in vivo. Even though double stranded nucleotides are generally more stable, siRNA is still extremely susceptible to enzymatic degradation in biological settings (Layzer et al., In vivo activity of nuclease-resistant siRNAs. RNA, 2004, 10(5):765-71). Also, due to its small size, siRNA is readily eliminated from the body through glomerular filtration. In many animal studies, renal clearance of siRNA correlated to higher uptake activities fan de Water et al., Intravenously administered short interfering RNA accumulates in the kidney and selectively suppresses gene function in renal proximal tubules, Drug Metab Dispos, 2006, 34(8): 1:393 -7). Clearance occurs in the kidney where the phagocytes aggregate the siRNA by using the serum proteins. The nuclease degradation of siRNA can occur by the reticuloendothelial system (RES). Finally, the negatively charged and hydrophilic small RNA structures have minimal interaction with the cell membrane (which is also negatively charged), and therefore, the cellular internalization is negligible.
Several delivery systems have been evaluated to improve the efficiency of siRNA, which include polymers, lipids, liposomes, and carbon nanotubes (Liu et al., SiRNA delivery systems based on neutral cross-linked dendrimers. Bioconjug Chem, 2012, 23(2):174-83; Chabot et al., Targeted electro-delivery of oligonucleotides for RNA interference: siRNA and antimiR. Adv Drug Deliv Rev, 2015, 81:161-8; de Fougerolles et al., Interfering with disease: a progress report on siRNA-based therapeutics. NatRev Drug Discov, 2007, 6(6):443-53; Golzio et al., In vivo gene silencing in solid tumors by targeted electrically mediated siRNA delivery. Gene Ther, 2007, 14(9):752-9; Oh et al., siRNA delivery systems for cancer treatment. Adv Drug Deliv Rev, 2009, 61(10):850-62), However, the majority of the delivery systems exhibited significant cytotoxicity and/or demonstrated limited in vivo and clinical potential. For example, poly(ethyleneimine) (PEI) is a commonly used polymeric carrier for siRNA delivery. The use of PEI facilitates the endosomal release of siRNA in the cellular cytoplasm. However, PEI induces toxicity contributes to low transfection efficiency (Guo et al., Engineering RNA for targeted siRNA delivery and medical application. Adv Drug Deliv Rev, 2010, 62(6):650-66).
Because of the great therapeutic potential of nucleic acids, developing efficient delivery systems is highly demanded in clinical investigations. FDA recently approved Onpattro™ (siRNA/lipid complex) for the treatment of hATTR amyloidosis. The delivery systems should be biodegradable to minimize the toxicity in the cytoplasm. Cell-penetrating peptides (CPPs) have drawn significant attention in the drug delivery field. CPPS take advantage of their unpatrolled properties like biocompatibility and cell penetration ability to pass the plasma membrane or through endocytic pathways. The intrinsic property of CPPs to deliver therapeutic molecules (nucleic acids, drugs, imaging agents) to cells and tissues in a nontoxic manner has indicated that they may be beneficial for enhancing the cellular delivery of drugs and diagnostic agents with limited cellular uptake. The modification of CPPs is required to act as delivery vectors for nucleic acid delivery to enhance interaction with the nucleic acid and to minimize the degradation and mediate the efficient cellular uptake. Often, the modification of CPPs can occur by attaching hydrophilic and hydrophobic lipid tails via linker residues. Attachment of lipids to the CPPs has been shown to facilitate membrane interactions and improve cellular uptake (Hafez et al., On the mechanism whereby cationic lipids promote intracellular delivery of poly nucleic acids. Gene Ther, 2001, 8(15):1188-96).
Cyclic peptides containing alternate tryptophan and arginine residues [WR]5 (SEQ ID NO.: 1) and [WR]4(SEQ ID NO.: 2) (
The delivery of siRNA complexes has been improved by targeting surface biomarker exclusively overexpressed on the disease's cells. One such example is targeting the 60 vβ3 integrin receptor in the angiogenic blood cells and tumor using linear or cyclic RGD peptide to improve the delivery of siRNA to the cancerous cells (Fu et al., RGD peptide-based non-viral gene delivery vectors targeting integrin αv β3 for cancer therapy. J Drug Target 2019, 27:1-11).
Thus, there is still a need for compositions and methods that provide efficient and non-toxic delivery of siRNAs to living cells. The compositions and methods disclosed herein addresses these and other needs.
Disclosed herein is a peptide-based systems containing hydrophobic amino acids (e.g., tryptophan), charged amino acids (e.g., arginine), and/or sulfur-containing amino acids (e.g., cysteine), which can be used either alone or in combination with nanoparticles (e.g., gold or silver nanoparticles) for siRNA delivery into living cells. Some of the peptides contain a disulfide bridge for cyclization and two chains of hydrophobic tryptophan residues. The amphiphilic cyclic and linear peptides and the corresponding peptide-capped gold nanoparticles were compared for siRNA delivery (Nasrolahi Shirazi et al., 2014). The rationale of this design is based on the data that a cyclic peptide [WR]5 (SEQ ID NO.: 1) containing alternating tryptophan (W) and arginine (R) residues (e.g., WRWR ((SEQ ID NO.: 3)) significantly enhanced the cellular uptake of negatively charged phosphopeptides and siRNA (Nasrolahi Shirazi et al., 2014).
Peptide carriers containing tryptophan, arginine, and/or cysteine for binding with siRNA, protection against enzymatic degradation, cellular internalization, and silencing are disclosed. The peptides and their corresponding gold nanoparticles were used in different ratios with siRNA for comparative studies in cancer cells. Furthermore, the effect of hydrophobic modification of the formulation of each peptide/siRNA complex was also evaluated. The size and surface electrical charge were evaluated for the complexes formed via ionic interaction between the peptides and siRNA to confirm complex formation and neutralization of siRNA negative charge and to evaluate the physical characteristics of the complex. The binding affinity of the peptides to siRNA were analyzed to determine the effect of peptide and the size of the conjugate on the inter-ionic interaction. The ability of the peptides in protecting siRNA against early enzymatic degradation was investigated by exposing the complexes to fetal bovine serum, and the toxicity of the peptides was explored in different human cancer cell lines. Finally, the efficiency of the peptides in internalizing siRNA into different human cancer cells was studied, which was confirmed by silencing efficiency for specific proteins.
Although embodiments that incorporate siRNAs are described at length, peptide and/or peptide-capped nanoparticles are useful for the delivery of other species of nucleic acid (e.g., ssDNA, dsDNA, DNA:RNA hybrids, etc.) as well as other negatively charged polymer species. As such, compositions and methods disclosed herein can be utilized in gene silencing, transient expression, and transient or permanent genetic modification in living cells,
Delivery of short interfering RNAs (siRNAs) remains a major challenge in the development of RNA interference therapeutics. siRNA is a class of double-stranded RNA molecules that are about 20-25 base pairs. An optimal siRNA delivery system for in vivo use must facilitate cellular uptake and enhance its activity. Second, it must direct the siRNA toward the target tissue effectively.
Compositions disclosed herein can include linear, cyclic, and/or hybrid linear/cyclic peptides containing natural or non-natural positively-charged amino acids, hydrophobic residues, and/or cysteine residues for use as siRNA delivery systems. Disclosed herein are four classes of peptides, which can include the addition of cysteine residues that can form disulfide bonds, modifications, such as the substitution of L-amino acid with D-amino acids to avoid proteolytic enzymes, the substitution of positively charge arginine or hydrophobic residues with non-natural amino acids, have significantly higher stability, loading efficiency, less toxicity, and effective silencing. Furthermore, the disulfide bridge can be reduced in the presence of glutathione, possibly releasing encapsulated siRNA in the peptide/siRNA complex.
Various objects, features, aspects, and advantages of the subject matter disclosed herein will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawing figures in which like numerals represent like components.
One should appreciate that the disclosed techniques provide many advantageous technical effects, including the efficient and direct introduction of nucleic acids (such as siRNA) into living cells.
Although each embodiment represents a single combination of inventive elements, the disclosed subject matter is considered to include all possible combinations of the disclosed elements. Thus, if one embodiment comprises elements A, B, and C, and a second embodiment comprises elements B and D, then other remaining combinations of A, B, C, or D, are considered disclosed and contemplated even if not explicitly disclosed.
In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable, The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.
Throughout the description and claims of this specification the word “comprise” and other forms of the word, such as “comprising” and “comprises,” means including but not limited to, and is not intended to exclude, for example, other additives, components, integers, or steps.
As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur and that the description includes instances where said event or circumstance occurs and instances where it does not.
The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
The term “hybrid peptide” is used to represent a peptide, which consists of a combination of linear and cyclic peptides. The peptide library was screened for their biophysiochemical properties, such as binding affinity for siRNA, particle size and surface charges of peptide/siRNA complex, and the stability of the complex in the presence of serum, and the siRNA release profile to establish a structure-function relationship.
Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified, thus fulfilling the written description of all Markush groups used in the appended claims.
Compositions
The first class of peptides contains positively charged amino acids (e.g.) arginine and hydrophobic amino acids (e.g., tryptophan residues) in an alternating fashion, where the peptide is cyclized through a disulfide linkage of two cysteine residues positioned at the IV- and C-termini of a linear precursor peptide (
Class 1 includes [C(RW)nC]; wherein n=1-10. The increase in positive charge density and hydrophobicity will enhance optimal interaction with siRNA by modulating R and W residues (alternate or R on one side and W on opposite side) between the disulfide bridges. To demonstrate the role of the disulfide bridge in the release of nucleic acid from the peptide/siRNA complexes, the linear and amide derivatives of peptides (replacement of both C amino acids with glutamic acid (E) and lysine (K) residues to form amide bridge) were also synthesized.
A second class of compounds (
A third class of peptides (
This class of peptides Wx[CRyC]Wx or (Rx[CWyC]Rx where x=1-10, y=1-10) is based on the lead peptide W4[CR5C]W4(SEQ ID NO.: 14). The optimal balance of hydrophobicity was achieved with optimizing linear chains of residues and R residues in the cyclic component in this class of peptides.
Alternatively, the corresponding linear peptides (W)n(KRnK)(W)n and (W)n(Rn)(W)n can be included in this group. The number of positively charged (e.g., arginine) and hydrophobic (e.g., tryptophan) residues can range from 1 to 10.
The fourth class of compounds composed of linear and cyclic peptides containing positively-charged residues such as arginine and hydrophobic residues such as tryptophan separated from each other by beta alanine (W4(β-alanine)x-Rn-(β-alanine)x where n=1-10, and x=1-5. Examples of compounds in this group are [W4-βAla-R4-βAla] (SEQ ID NO.: 5) and (W4-βAla-R4-βAla) ((SEQ ID NO.: 6)).
Fourth classes of compounds can be used alone for nucleic acid delivery or be used for the generation of peptide-capped gold nanoparticles that are used for nucleic acid delivery.
The peptides (see examples in Table 1) were synthesized using Fmoc/tBu solid-phase synthesis. In brief, the linear peptides were assembled on trityl resin. The amino acids in the sequence were conjugated using Fmoc-amino acid building blocks in the presence of 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), and diisopropylethylamine (DIPEA) in dimethylformamide (DMF). After each coupling, the Fmoc deprotection performed using 20% (v/v) piperidine in DMF. The progress of the reaction was monitored by matrix-assisted laser desorption/ionization (MALI) time-of-flight (TOF) analyzer. The resultant peptides were cleaved from the resin, followed by purification and lyophilization. The linear peptides containing cysteine residues were oxidized in the presence of 10% DMSO/Water to introduce disulfide bridges. The amide bridged derivative was synthesized by using orthogonal protecting K and E residues that are selectively deprotected on a solid support to form an amide bridge in the side chains followed by complete deprotection and purification.
Linear, cyclic, hybrid cyclic-linear peptides were synthesized that have hydrophobic positively-charged, and/or cysteine residues as the amino acid sequence in its basic structure. Inventors varied the amino acid constituents in the structure to determine the effectiveness of derived compounds in siRNA binding and delivery and to establish a structure-activity relationship. The strategy was to vary net hydrophobicity and the positive charges of derived compounds based on structure-activity relationships and evaluate siRNA binding, delivery, and silencing potentials. The rationale of current studies was that cancer cells have a higher percentage of glutathione relative to normal cells. Accordingly, disulfide bonds can be reduced in cancer cells to a significantly higher degree than normal cells, providing selective delivery of siRNA to cancer cells.
Compounds disclosed herein represent a new class of transfection delivery agents. The structures of these series of compounds are different than those of current delivery agents. The used amino acids, peptide sequence, examples of their structures, in vitro siRNA binding affinity, cytotoxicity, siRNA delivery, and silencing potential are summarized herein.
Preferred compound(s) could be used as a stand-alone or to be used in generating peptide-capped gold or silver nanoparticles for siRNA delivery. These peptides can be used in combination with siRNA. in different ratios. Alternatively, peptide-capped nanoparticles of these peptides can be combined with siRNA. The peptides can be used alone or in combination with nanoparticles and peptide-capped nanoparticles. Examples of suitable nanoparticles include metal nanoparticles (e.g., gold and/or silver nanoparticles) that can be used in combination with peptides to improve the delivery of siRNA. Cell-penetrating peptide-capped nanoparticles with antimicrobial properties will be preferentially taken up by the cells where they gradually release their cargo siRNA, resulting in sustained local silencing effect by a two-pronged mechanism without causing significant toxicity to normal cells. Peptides-capped metal nanoparticles have cell-penetrating properties as membrane permeabilizers. Cell-penetrating peptides can entrap siRNA. and enhance the uptake of siRNA across the membrane when they cap the metal nanoparticles.
Compounds disclosed herein represent a new class of nucleic delivery agents. The structures of these series of compounds are different than those of current delivery agents. The used amino acids, peptide sequence, examples of their structures, in vitro siRNA binding affinity, cytotoxicity, siRNA delivery, and silencing potential are summarized herein.
Amino acids: Examples of suitable positively-charged amino acids in the linear and cyclic peptides are L-arginine, L-lysine, L-histidine, D-histidine, D-arginine, D-lysine. Furthermore, positively-charged amino acids are L- or D-arginine and L- or D-lysine, ornithine, L- or D-histidine residues with shorter or longer side chains (e.g., C3-Arginine (Agp), C4-Arginine (Agb)), diaminopropionic acid (Dap) and diaminobutyric acid (Dab), amino acids containing free side-chain amino or guanidine groups, and modified arginine and lysine residues.
Examples of suitable hydrophobic residues in the linear and cyclic peptides are L-tryptophan, D-tryptophan, L-phenylalanine, D-phenylalanine, L-isoleucine, D-isoleucine, p-phenyl-L-phenylalanine (Bip), 3,3-diphenyl-L-alanine (Dip), 3(2-naphthyl)-L-alanine (NaI), 6-amino-2-naphthoic acid, 3-amino-2-naphthoic acid, 1,2,3,4-tetrahydronorharmane-3-carboxylic acid, 1,2,3,4-tetrahydro-3-isoquinolinecarboxylic acid (Tic-OH), 1,2,3,4-tetrahydro-3-isoquinolinecarboxylic acid, modified d- or 1-tryptophan residues like N-alkyl or N-aryl tryptophan, substituted d- or L-tryptophan residues (e.g., 5-hydroxy-L-tryptophan, 5-methoxy-L-tryptophan, 6-chloro-L-tryptophan), other N-heteroaromatic and hydrophobic amino acids, and fatty amino acids (e.g., NH2-(CH2),COOH, where x=1-20).
Sequence: A preferred sequence of these peptides includes linear (C(XY)nC) or cyclic [(C(XY)nC)] (through disulfide bridge or N- to C-terminal or both), linear [C(X)n(Y)nC)] or cyclic [(C(X)n(Y)n)C)] (through disulfide bridge or N- to C-terminal or both), linear (Y)nCXnC(Y)n, (X)nCYnC(X)n or hybrid cyclic-linear (Y)n[CXnC](Y)n or hybrid cyclic-linear (X)n[CYnC](X)n or hybrid cyclic-linear (Y)n[KXnK](Y)n or hybrid cyclic-linear (X)n[KYnK](X)n or linear (X)n(KYnK)(X)nor linear (X)n(Yn)(X)n, wherein X is a positively-charged amino acid, Y is a hydrophobic residue or [Xn-(β-alanaine)x-Yn-(β-alanaine)x], wherein n=1-10. Other amino acids can be inserted between positively-charged, between hydrophobic residues, or between positively-charged and hydrophobic residues while multiple positively-charged residues or multiple hydrophobic amino acids are next to each other, creating a positively-charged component on one side and a hydrophobic component on the other side. Similar or different positively charged or hydrophobic residues may be in the same peptide. In other words, positively charged amino acids can be the same or different. Similarly, hydrophobic amino acids in the same sequence can be the same or different. The peptides can have hybrid structures with cyclic peptides contain positively-charged residues or hydrophobic residues attached to two linear hydrophobic or positively-charged residues. Some of the sequences are shown in Table 1 and
In some examples, peptides (cyclic [CR4W4C] (SEQ NO.: 16) and hybrid W:4[CR5C]W4) (SEQ ID NO.: 52) are disclosed; and other peptides in each group are inexpensive, easy-to-use, nontoxic, serum-stable, and efficient transfecting agent for all mammalian cells and in vitro nucleic acid delivery.
In one aspect, disclosed are CPs and hybrid cyclic-linear peptides (HCLPs). The concept of using a cyclic peptide-based transfection agent will provide intrinsic property associated with a conformationally constrained structure, such as selectivity to a class of siRNA or cell line, stability, and protection to siRNA from nuclease and serum, and low toxicity. CPs and HCLPs synthesized from natural L-amino acid will be biodegradable, leading to minimum toxicity as compared to lipid or polymer-based transfection agents. The CPs-based transfection agent will be economical as compared to lipid or polymer-based transfecting agents. Structural design of the CPs in the proposed study originated after extensive modification of previously developed classes of CPs containing W and R residues in the cyclic ring ([WR]n; n=4,5) or R/W residues in the cyclic ring followed by a linear chain of R/W residues (called HCLPs: [R5K]W5 (SEQ ID NO.: 92) and [R6K]W6(SEQ ID NO.: 93)) or R residues in the cyclic ring containing chain of fatty acids ([(R5(K-C16)2], [R5(K-C18)2]) that demonstrate diverse applications in the non-covalent delivery of siRNA., By controlling and balancing inter-residue electrostatic and hydrophobic interactions with the siRNA. molecules, several classes of CPs and HCLPs were synthesized and evaluated with variable cellularization and efficiency in siRNA delivery. Efforts in structural modification provided several lead peptides (cyclic [CR4W4C] (SEQ ID NO.: 16) and hybrid W4[CR5C]W4(SEQ ID NO.: 52)), which demonstrated high transfection efficiency without toxicity, serum stability as compared to commercial agent lipofectamine. Lead peptide [CR4W4C] (SEQ ID NO.: 16) is cyclized through intramolecular disulfide bridge between cysteine residues, which seems important in the release of siRNA from peptide-siRNA. complex in the cytoplasm after being reduced with glutathione. The reduction of the disulfide bridge provides conformational changes in the peptide-siRNA. complex, which modulates the release of siRNA. Furthermore, these peptides are internalized independently of endocytic pathways, which is a significant and distinct property as compared to other drug delivery platforms. The unique and intrinsic property of lead peptides to spontaneously translocate across bilayers is distinct from the behavior of well-known, highly cationic CPPs, such as TAT and Arg9, which do not translocate across synthetic bilayers and instead enter cells primarily by endocytosis. Endosomal entrapment limits nucleic acid transfection efficiency significantly, as the nucleic acids are trapped in endocytic compartments and cannot reach their cytoplasmic or nuclear targets. The other significant advantage of the proposed structures is their high stability and low toxicity. Highly cationic CPPs preferentially interact with particular cell types, have limited serum half-life, show toxicity, and require 9-15 residues of R for efficient cargo delivery. Compared to linear CPPs, which are susceptible to hydrolysis by endogenous peptidases, cyclic peptides are enzymatically more stable and non-toxic. CPs and HCLPs can be used for different nucleic acids (including clustered regularly interspaced short palindromic repeats or CRISPR). Unlike many commercially available transfection agents that could not even be considered for in vivo use, the versatile structure of the proposed peptides offers the possibility of future modifications, e.g., incorporation into gold nanoparticles, conjugation with polyethylene glycol (PEG), and/or targeting moieties for stealth properties and active targeting, respectively, which create clinically relevant delivery systems.
The peptides are amphipathic in nature and conformationally constrained due to the intramolecular disulfide bridge between cysteine residues (
The structural modifications in the lead peptides were used to optimize them as efficient vectors for cellular delivery of siRNA. through enhancing their interactions with negatively charged siRNA and providing them protection from the nucleases, and mediating the efficient cellular uptake.
The balance of amphipathic character, density, and position of positive charges and hydrophobic moieties in the conformationally constrained structures of peptides generated an efficient transfecting agent.
Classes 1 and 2 CPs contain disulfide and amide bridges and were developed to generate a conformationally constrained structure. Cyclic peptides are found to be more resistant to cellular metabolic enzymes, such as proteases and nucleases. Furthermore, the higher level of glutathione at the cellular level in various disease stages will facilitate the reduction of the disulfide bridge of peptide in the CPs containing disulfide bridge and thus changing the constrained conformation of the peptide to a flexible structure (a linear form of peptide), releasing the siRNA. Changes in the conformation of peptides in the peptide/siRNA complex will help in releasing the cargo inside the cytoplasm.
The data indicated that the lead cyclic [CR4W4C] (SEQ ID NO.: 16) and hybrid W41[CR5C]W4 (SEQ ID NO.: 52) peptides containing disulfide bridge demonstrated potential applications as transfecting agents. Classes 1 and 2 peptides explore both the role of the disulfide bridge and constrained cyclic structure in developing optimal transfecting agents.
Class 3 represents HCLP analogs, composed of hydrophobic linear chains attached to a cyclic peptide containing positively charged R residues or o linear chains of positively charged residues on a cyclic peptide of W residues.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices, and/or methods claimed herein are made and evaluated and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, the temperature is in ° C. or is at ambient temperature, and pressure is at or near atmospheric.
The peptides can be used as pegylated peptides with or without targeting moieties such as RGD, folic acid, transferrin, antibody for nucleic acid delivery.
All amino acid building blocks and preloaded amino acid on the resin used in this study were purchased from AAPPTEC. Other reagents, chemicals, and solvents were procured from Sigma-Aldrich. The final compounds used in further studies were purified with reversed-phase High-performance liquid chromatography from Shimadzu (LC-20AP) using a binary gradient system of acetonitrile 0.1% TFA and water 0.1% TEA and a reversed-phase preparative column (X Bridge BEH130 Prep C18, 10 μm 18×250 μm Waters, Inc). The chemical structure of linear, cyclic, and hybrid cyclic-linear peptides, intermediates, and final products were characterized by high-resolution MALDI-TOF (GT-0264) from Broker Inc.
General method for the synthesis of linear peptides. The synthesis of linear peptides was performed by Fmoc/tBu solid-phase peptide synthesis method. All the peptides were synthesized using Fmoc solid-phase peptide synthesis using appropriate resin and Fmoc-protected amino acids.
Protected amino acid-2-Cl trityl resin and Fmoc protected amino acids were used as the building blocks for the synthesis peptides on a scale of 0.3 mmol. 2-H-benzotriazol -1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU)/N,N-diisopropylethylamine (DIPEA) were used as coupling and activating reagents, respectively. Piperidine in N,N-dimethylformamide (DMF) (20%, v/v) was used for Fmoc deprotection. The resultant peptides were cleaved from the resin, and all protecting groups were removed using a cleavage cocktail of TFA/anisole/EDT/thioanisole (90:5:3:2, v/v/v/v) for 3 h. Crude products were precipitated by the addition of cold diethyl ether purified by reverse phase HPLC using a gradient of 0-90% acetonitrile (0.1% TFA) and water (0.1% TFA) over 60 min with C-18 column. Purified peptides were lyophilized to yield a white powder. The chemical structures of all synthesized peptides were elucidated using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectroscopy with α-cyano hydroxycinnamic acid (CHCA) as a matrix.
MALDI-TOF (m/z) for C(RW)4C (SEQ ID NO.: 20) [C74H100N26O11S2]: calcd,1592.7506; found, 1592.7797 []−; MALDI-TOF (m/z) for C(RW)5C (SEQ ID NO.: 94) [C91H122N32O13S2]: calcd,1934.9311; found, 1934.2925 [M]+; MALDI-TOF (m/z) for CR4C(SEQ ID NO.: 16) [C74H100N26O11S2]: calcd, 1592.7506; found, 1593.3369 [M+H]+; MALDI-TOF (m/z) for CR5W5C (SEQ ID NO.: 56) [C91H122N32O13S2]: calcd,1934,9311; found, 1936.445 [M+2H]+; MALDI-TOF (m/z) for R4CW4C (SEQ ID NO.: 95) [C74H100N26O11S2]: calcd, 1592.7506; found, 1593.957 [M+H]+; MALDI-TOF (m/z) for W4CR4C (SEQ ID NO.: 96) [C74H100N26O11S2]: calcd,1592.7506; found, 1593,160 [M+H]+; MALDI-TOF (m/z) for W2CR5CW2 (SEQ ID NO.: 97) [C80H112N30O12S2]: calcd, 1748.8517; found, 1749.475 [M+H]+; MALDI-TOF (m/z) for W3CR5CW3 (SEQ ID NO.: 98) [C102H132N34O14S2]: calcd, 2121.0104; found, 2122.159 [M+H]+; MALDI-TOF (m/z) for W4CR5CW4(SEQ ID NO.: 99) [C124H153N39O15D2]: calcd, 2493.1850; found, 2494.2889 [M+H]+.
Synthesis of cyclic peptides through N- to C-cyclization. Amino acid loaded on trityl resin and Fmoc-amino acid building blocks were used for the synthesis on a scale of 0.3 mmol. HBTU/DIPEA was used as coupling and activating reagents, respectively. Piperidine DMF (20% v/v) was used for Fmoc deprotection. The side-chain protected peptides were detached from the resin by TFE/acetic acid/DCM [2:1:7 (v/v/v)] then subjected to cyclization using 1-hydroxy-7-azabenzotriazole (HOAT) and N,N′-diisopropylcarbodiimide (DIC) in an anhydrous DMF/DCM mixture overnight. All protecting group were removed with cleavage cocktail of TFA/anisole/thioanisole (90:2:5 v/v/v) for 3 h. The crude products were precipitated by the addition of cold diethyl ether purified using reverse-phase HPLC using a gradient of 0-90% acetonitrile (0.1% TFA) and water (0.1% TFA) over 60 min with C-18 column. The purified peptide was lyophilized to yield a white powder (100 mg). The chemical structure of all synthesized peptides was elucidated using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometer.
MALDI-TOF (m/z) for [R4CW4C] (SEQ ID NO.: 95) [C74H98N26O10S2]: calcd, 1574.7401; found, 1575.578 [M+H+].
Synthesis of cyclic peptides cyclized through a disulfide bridge. About 30 mg of the linear peptide was dissolved in a 10% DMSO-H2O solution (150 ml). The reaction mixture was stirred for 24-72 h at room temperature in an open round-bottomed flask. The reaction mixture was injected directly in reverse phase HPLC using a gradient of 0-90% acetonitrile (0.1% TFA) and water (0.1% TFA) over 60 min with C-18 column. The purified peptide was lyophilized to yield a white powder (20 mg). The chemical structure of all synthesized peptides was elucidated using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometer.
MALDI-TOF (m/z) for ([C(WR)4C]
(SEQ ID NO.: 100) [C74H98N26O11S2]: calcd, 1590.7350; found, 1590,8999 [M]+.
MALDI-TOF (m/z) for [C(WR)5C]
(SEQ ID NO.: 101) [C91H120N32O13S2]: calcd,1932.9154; found, 1932.9976 [M]+.
MALDI-TOF (m/z) for
[C74H98N26O11S2]: calcd, 1590,7350; found. 1591.161 [M+H]+.
MALDI-TOF (m/z) for
[C91H120N32O13S2]: calcd, 1932.9154; found, 1937.6542 [M]+.
MALDI-TOF (m/z) for
[C74H96N26O10S2]: calcd, 1572.7244; found, 1573,148 [M+H]+.
MALDI-TOF (m/z) for R4[CW4C] [C74H98N26O11S2]: calcd, 1590,7350; found, 1590.873 [M]+.
MALDI-TOF (m/z) for W4[CR4C][C74H98N26O11S2]: calcd, 1590.7350; found, 1591.286 [M+H]+.
MALDI-TOF (m/z) for
[C80H110N30O12S2]: calcd, 1746.8410; found, 1746.994 [M]+.
MALDI-TOF (m/z) for
[C102H130N34O14S2]: calcd, 2118.9991; found, 2119.1252 [M]+.
MALDI-TOF (m/z) for
[C124H151N39O15S2]: calcd, 2491.1850, found, 2491.5856 [M]+.
MALDI-TOF (m/z) for (W)2[KR5K](W)2[C86H124N32O11]: calcd, 1781.0127; found, 1781.61 [M]+.
MALDI-TOF (m/z) for (W)[KR5K](W) [C63H104N28O9]: calcd, 1408.8541; found, 1410,0432 [M+H]+.
As representative examples, the synthesis of representative linear and cyclic peptides is shown in
Synthesis of Gold nanoparticles. In a typical synthesis, an aqueous solution of peptide and chloroauric acid were mixed in the equimolar ratio (1:1) (2 mM peptide solution and 2 mM chloroauric acid) and stirred at room temperature for 24 hours (
Synthesis of the pegylated cell-penetrating peptide (PEG-CPP). The solid-phase synthesis of the linear peptide sequence was conducted using the standard protocol mentioned before. The desired peptide sequence was assembled on rink amide resin. Orthogonal protected Cys(StBu) residues were incorporated to achieve on-resin sulfur bridge formation. N-Terminal of parent peptide was further extended by coupling 2-Fmoc aminoethoxy acetic acid as a spacer between the parent peptide and another cysteine residue Cys(Mmt), which was installed to pegylate the peptide by exploiting free side chain thiol functionality. The tent-butyl thiol groups were deprotected on resin using 3.8 mmol dithiothreitol (DTT) in 2.5 mL DMF with 0.25 mL DIEA. The reaction proceeded at 60° C. for 20 minutes. The resin was washed thoroughly with DMF and DCM. On-resin disulfide formation reaction was conducted using N-chlorosuccinimide (NCS, 2 eq.) in DMF Methoxytrityl group was deprotected selectively by repeatedly treating the peptidyl resin with 2% TFA in DCM. The resulted free thiol functionality was used for pegylation of the peptide by reacting with preactivated PEG (mPEG-OPSS; Orthopyridyl disulfide). For on-resin pegylation reaction, 2 eq. of preactivated PEG-SH was added to the peptidyl resin in DMF. The reaction mixture was kept for shaking at room temperature for around 14 hours (
Synthesis of pegylated targeting peptide (PEG-TP). Synthesis of pegylated targeting peptide was conducted on wang resin as represented in the scheme (
Synthesis of the final conjugate (TP-PEG-CPP). As depicted in the scheme, the final conjugate was synthesized by sulfur bridge formation between pegylated targeting peptide (PEG-TP) and cell-penetrating peptide (CPP). Purified pegylated targeting peptide (2 equiv.) having preactivated thiol was added to peptidyl resin in DMF. The reaction mixture was kept for shaking at room temperature for around 14 hours. Final conjugate was cleaved from the resin and purified by RP-HPLC and mass was confirmed by Q-TOF-LC-MS.
Complex formation with modified peptides and siRNA. The peptide/siRNA complexes were formed in different N/P ratios for the variety of the in vitro studies conducted according to the previously reported procedure (Mozaffari et al., 2019). The complexes formed spontaneously based on ion/ion interaction in normal saline. For each formulation, the required amount of scrambled siRNA stock solution (10 μM) was added to normal saline, and then, the calculated amount of peptide stock solution (1 mg/mL) was added to the solution. The mixture was gently mixed and was incubated at room temperature for 30 minutes to ensure complete complexation. The peptide:siRNA ratios used in complex formations are reported as weight/weight ratios, as well as nitrogen/phosphate (N/P) ratios. The N/P ratios were calculated using the following formula:
NP=(#of Moles peptide×# of nitrogens in each molecule of peptide) (#of Moles of siRNA×# of phosphate groups in each molecule)
All siRNAs used in this project contain 21-25 base pairs in each strand. The scrambled siRNA used in this set of experiments contains 23 base pairs.
Size and Surface Charges. The efficiency of siRNA carriers largely depends on the size and overall surface charge of the complex. Dynamic light scattering (DLS) was used to determine the size and ζ-potential at different N/P ratios to determine the optimal N/P ratio for a compact complex (50-200 nm) and slightly positive charge.
siRNA binding affinity. To evaluate binding affinity to siRNA, a study was designed based on the quantification of free siRNA using SYBR Green II. Peptides were mixed with siRNA with a wide range of N/P ratios (0.05-40), and the amount of unbound siRNA was quantified by the fluorescent signal of SYBR Green II. The required N/P ratio for 50% binding (BC50) was calculated for each peptide.
Serum Stability. In order to determine the capability of the complexes to protect the siRNA against enzymatic degradation, Inventors exposed different study groups to a diluted fetal bovine serum (FBS) solution, with “naked” siRNA as a positive control, and siRNA exposed to saline as a negative control according to the previously reported procedure (Mozaffari et al., 2019), and the results are summarized in
The binding affinity for [CR4W4C](SEQ ID NO.: 16) and W4[CR5C]W4(SEQ ID NO.: 52) showed that approximately 4 and 10 N/P ratios are required for 50% binding. While “naked” siRNA. was completely degraded in the presence of serum, both peptides were able to protect siRNA completely after 24 hours of exposure to the serum (as compared to siRNA exposed to saline as negative control) at an N/P ratio of 40 (
Protection of siRNA against Enzymatic Degradation. In order to study the capability of the fatty acid-conjugated linear and cyclic peptides to enhance the stability of siRNA in biological environments, samples of unprotected scrambled siRNA (positive control) and peptide/siRNA complexes at a different peptide:siRNA ratios were exposed to fetal bovine serum (FBS) solutions. After completion of complex formation for each formulation, each sample was added to a 25% v/v FBS solution in HBSS, and the mixture was incubated at 37° C. for 24 h. A sample of unprotected siRNA in HBSS was used as a negative control, representing 100% intact siRNA. After the incubation, complexes were dissociated using a 2:3 mixture of heparin (5% solution in normal saline) and EDTA (0.5 mM), and the samples were analyzed using a 1% agarose gel (with 1 μg/mL ethidium bromide) at 70 V for 20 minutes. UV illumination (Gel-Doc system, Bio-Rad; Hercules, CA) was used to visualize the gel, and the intensity of the bands (representing remaining intact siRNA) was quantified by Image J software.
Cylotoxicity Assays. The cytotoxicity of the peptides was evaluated in a panel of cancer and non-cancerous cell lines. Normal human breast (Hs 578Bst, ATCC™ HTR-125™), kidney (LLCP-K1, ATCC CRL-1392), prostate (RWPE-1; ATCC™ CRL-11609™), colon (CCD-33Co; ATCC™ CRL-1539™), heart (H9C2. ATCC CRL 1446) or endometrial (KC02-44D hTERT, ATCC™ SC-6000™) cell lines were exposed to a wide range of peptide/siRNA concentrations. Due to the popularity of siRNA silencing in cancer research, the toxicity of the same study groups were also evaluated in TNBC cells MDA-MB-231 (ATCC™ HTB-26™), MDA-MB-468 (ATCC™ HTB-132), ovarian SK-OV-3 (ATCC™ HTB-77™), colorectal ht-29 (ATCC™ HTB-38™), and prostate LNCaP (ATCC™ CRL-174™) cancer cell lines. Cell viability was evaluated by CCK assay after 48 hours of exposure. Neither of the peptides showed significant toxicity in the range of concentration or N/P ratio evaluated (
The human breast cancer cell line MDA-MB-231 was purchased from American Type Culture Collection (ATCC; Manassas, Va.). MDA-MB-231 was cultured in the DMEM medium. The media was supplemented with 10% (v/v) fetal bovine serum, 100 U/mL penicillin, and 100 μg/ml streptomycin. Cells were maintained in the normal condition of 37° C. and 5% CO2 under a humidified atmosphere and were sub-cultured when 80-100% confluent.
The &Tea of peptides on the viability of human cell lines was evaluated by exposing the MDA-MB-231 cell lines (
Cellular Uptake. Cellular uptake was quantified using flow cytometry and was evaluated in all the cell lines included in the cytotoxicity study. However, since this method did not confirm cytoplasmic accumulation, confocal microscopy was performed to confirm and visualize the cytoplasmic delivery. Free siRNA and Lipofectamine™ were used as negative and positive controls, respectively.
Cellular internalization was studied in MDA-MB-231 using flow cytometry and confocal microscopy. The percentage of cells identified as siRNA-positive for the complexes formed with two lead peptides using an N/P ratio of 20 was comparable to the percentage of cells achieved using commercially available Lipofectamine™ 2000 (˜63% vs. 72%;
The ability of peptides to deliver siRNA into the MDA-MB-231 cells was evaluated using FAM-labeled scrambled siRNA and analysis by flow cytometry (
The peptides showed significant siRNA uptake, which was significantly higher at a peptide:siRNA ratio of 20:1 and 40:1 after 24 h incubation. The mean fluorescence results and positive fluorescence cells confirmed a similar pattern. The addition of PEGylated peptide (25%) in the native peptide solution enhances the uptake of siRNA.
The efficiency of siRNA uptake in this cell line was also confirmed with confocal microscopy images (
The siRNA silencing efficiency were also evaluated in the same cells targeting signal transducer and activator of transcription 3 (STAT3) as a model protein. STAT3 is a well-studied protein with a central role in JAK2/STAT3 pathway and cancer cell proliferation and survival. The expression level of targeted protein was assessed using Western Blot, which showed comparable silencing efficiency of lead peptides to Lipofectamine™ 2000 (
Methodology for Cell-Based Assays. Cell Lines: The human breast cancer cell line MDA-MB-231 was purchased from American Type Culture Collection (ATCC; Manassas, Va.). MDA-MB-231 was cultured in the DMEM medium. The media was supplemented with 10% (v/v) fetal bovine serum, 100 U/mL penicillin, and 100 μg/ml streptomycin. Cells were maintained in the normal condition of 37° C. and 5% CO2 under a humidified atmosphere and were sub-cultured when 80-100% confluent.
Toxicity of the Peptide/siRNA Complexes: The safety profile of the peptides was evaluated in two different human cancer cell lines, by exposing the cells to the peptides alone and in peptide/siRNA complexes according to the previously reported procedure (Mozaffari et al., 2019). Confluent cultures (≈800,000 cells/mL) of MDA-MB-231 cancer cell lines were seeded in 96-well plates. After 24 h, cells were exposed to different concentrations of peptide solutions or peptide/siRNA complexes. The final concentration of scrambled siRNA was 36 nM in all cytotoxicity studies performed with peptide/siRNA complexes. The cells were then incubated for 48 hours at 37° C. with controlled CO2 and humidity. A standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed after the incubation period to determine the cell viability as a percentage of untreated (NT) cells.
Internalization of siRNA into Human Cancer Cell Lines: The capability of peptides in internalizing siRNA into human cells was evaluated using FAM-labeled scrambled siRNA and flow cytometry according to the previously reported procedure (Mozaffari et al., 2019). MDA-MB-231 cells were used for these studies and were seeded in 24-well plates (≈200,000 cells per well). After the addition of the siRNA complexes to cell culture media, cells were incubated at 37° C. and standard growth conditions for 24 hours. After the incubation period, and for flow cytometry studies, cells were washed with clear HBSS (x2), trypsinized, and fixed using 3.7% formaldehyde solution. Suspended cells were analyzed with a BD-FACSVerse (BD Biosciences; San Jose, Calif.) using the FITC channel to quantify cell-associated fluorescence. After each flow cytometry analysis, the percentage of cells with fluorescence signal and the mean fluorescence of the cell population were calculated based on the calibration of the signal gated with non-treated cells (as the negative control), so that the auto-fluorescence would be ≈1% of the population.
Gene Silencing. Signal Transducer and Activator of Transcription 3 (STAT3) protein was selected as a model protein to demonstrate the in vitro silencing efficiency of the designed modified peptides. STAT3 has been shown to play a major role in the proliferation and survival of different types of cancer, including breast cancer, and may even be involved in resistance against molecularly targeted drugs (Bousoik “Do Inventors Know Jack” About JAK? A Closer Look at JAK/STAT Signaling Pathway. Front Oncol, 2018, 8:287). Along with Janus Kinase 2 (JAK2), STAT3 is among the major proteins involved in this inter-pathway crosstalk, and latest reports have led to the elucidation of a key role of the JAK/STAT signaling pathway in the development, proliferation, differentiation, and survival of cancer cell, and in fact, Vogelstein et at have included JAK/STAT pathway among 12 core cancer pathways (Cancer genome landscapes. Science, 2013, 339:1546-58). The effect of STAT3 activation on Ras and PI3K/Akt pathways and the connections of JAK2 to PI3K and ERK pathways are examples of these inter-pathway cross-talks. Several peptides when used with siRNA, exhibited significant silencing of Stat3 similar to or better than lipofectamine (
Protein Quantification (Western Blot). The expression of the targeted protein was analyzed by western blot. Cell lysates were prepared according to the standard protocol using RIPA buffer. Briefly, cells exposed to siRNA complexes were collected by trypsinization after 48 h of exposure and were centrifuged at 600-800 RPM for 5 min. The supernatant was discarded, and the cell pellet was washed three times with ice-cold PBS. The pellet was resuspended in RIPA buffer (100 μL of buffer for 25 μL of cell pellet), and the cell lysates were then incubated on ice for one hour, during which the tubes were sonicated for 3 min every 10 min. The tubes were then centrifuged for 15 min at 12,000×g (at 4° C.). Microtubes were pre-cooled to transfer the supernatant, and total protein concentration was determined using BSA assay. Briefly, 200 μL of work reagent (50:1 A:B) was added to 25 μL of standard and unknown samples in triplicate into a 96-well plate, and the plate was mixed on a plate shaker for 30 s. The plate was then incubated at 37° C. and 5% CO2 for 30 min. The absorbance was measured at 562 nm using SpectraMAX M5 microplate reader. Protein (25 μg) was loaded per well in a 10% Mini-PROTEAN™ TGX Stain-Free™ Protein gel using electrophoresis buffer (0.192. M glycine, 25 mM Tris, 0.1% SDS), and the electrophoresis was run for 60 min with 100 V. The gel was then transferred onto a Trans-Blot™ Turbo™ Mini PVDF membrane (Catalog no. 1704156). Membranes were blocked in BSA 5% for 3 h, and then incubated overnight (at 4° C.) with the primary anti-body (1:1000 in TBS-T). The membrane was then washed with TBS-T three times (5 min each time) and was subsequently incubated with the secondary HRP-linked antibody (1:1000 in TBS-T) for 1 h, followed by the washing steps. Detection was done by ECL Detect Kit using CherniDoc imager (Bio-Rad).
The peptide/plasmid complexes formed with different peptides were able to effectively transfect HEK293 cells with Green Fluorescence Protein (GFP)-expressing plasmid with no apparent toxicity (compared to commercially available Lipofectamine, which causes cell death and aggregation in the culture media (
It should be apparent to those skilled in the art that many more modifications besides those already described are possible without departing from the concepts herein. The disclosed subject matter, therefore, is not to be restricted except in the spirit of the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms “comprises” and “including” should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced. Where the specification claims refer to at least one of something selected from the group consisting of A, B, C . . . , and N, the text should be interpreted as requiring only one element from the group, not A plus N, or B plus N, etc.
This application claims the benefit of priority to U.S. Provisional Application No. 62/953,365, filed Dec. 24. 2019, and No. 63/013,843, filed Apr. 22, 2020, the disclosures of which are incorporated by reference herein in their entireties.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/067036 | 12/24/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63013843 | Apr 2020 | US | |
| 62953365 | Dec 2019 | US |
