Modifying peptide/lipid-associated nucleic acids (PLANAs) for CRISPR/Cas9 ribonucleoprotein delivery.

With the first reports on the possibility of genome editing by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas)9 surfacing in 2005, the enthusiasm for protein silencing via nucleic acid delivery experienced a resurgence following a period of diminished enthusiasm due to challenges in delivering small interfering RNAs (siRNA), especially in vivo. However, delivering the components necessary for this approach into the nucleus is challenging, maybe even more than the cytoplasmic delivery of siRNA. We previously reported the birth of peptide/lipid-associated nucleic acids (PLANAs) for siRNA delivery. This project was designed to investigate the efficiency of these nanoparticles for in vitro delivery of CRISPR/Cas9 ribonucleoproteins. Our initial experiments indicated higher toxicity for PLANAs with the more efficient reverse transfection method. Therefore, polyethylene glycol (PEG) was added to the composition for PEGylation of the nanoparticles by partially replacing two of the lipid components with the PEG-conjugated counterparts. The results indicated a more significant reduction in the toxicity of the nanoparticle, less compromise in encapsulation efficiency and more PEGylation of the surface of the nanoparticles using DOPE-PEG2000 at 50 % replacement of the naïve lipid. The cell internalization and transfection efficiency showed a comparable efficiency for the PEGylated and non-PEGylated PLANAs and the commercially available Lipofectamine™ CRISPRMAX™. Next Generation Sequencing of the cloned cells showed a variety of indels in the transfected cell population. Overall, our results indicate the efficiency and safety of PEGylated PLANAs for in vitro transfection with CRISPR/Cas9 ribonucleoproteins. PEGylation has been studied extensively for in vivo delivery, and PEGylated PLANAs will be candidates for future in vivo studies.

Hydrophobicity drives the cellular uptake of short cationic peptide ligands.

Short cationic linear peptide analogs (LPAs, prepared as Arg-C( n )-Arg-C( n )-Lys, where C( n ) represents an alkyl linkage with n = 4, 7 or 11) were synthesized and tested in human breast carcinoma BT-20 and CCRF-CEM leukemia cells for their application as targeting ligands. With constant LPA charge (+4), increasing the alkyl linkage increases the hydrophobic/hydrophilic balance and provides a systematic means of examining combined electrostatic and hydrophobic peptide-membrane interactions. Fluorescently conjugated LPA-C(11) (F-LPA-C(11)) demonstrated significant uptake, whereas there was negligible uptake of the shorter LPAs. By varying temperature (4°C and 37°C) and cell type, the results suggest that LPA-C(11) internalization is nonendocytic and nonspecific. The effect of LPA binding on the phase behavior, structure, and permeability of model membranes composed of dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylserine (DPPC/DPPS, 85/15) was studied using differential scanning calorimetry (DSC), cryogenic transmission electron microscopy (cryo-TEM), and fluorescence leakage studies to gain insight into the LPA uptake mechanism. While all LPAs led to phase separation, LPA-C(11), possessing the longest alkyl linkage, was able to penetrate into the bilayer and caused holes to form, which led to membrane disintegration. This was confirmed by rapid and complete dye release by LPA-C(11). We propose that LPA-C(11) achieves uptake by anchoring to the membrane via hydrophobicity and forming transient membrane voids. LPAs may be advantageous as drug transporter ligands because they are small, water soluble, and easy to prepare.

Synthesis, antiviral and contraceptive activities of nucleoside-sodium cellulose sulfate acetate and succinate conjugates.

Chemical conjugates between sodium cellulose sulfate (CS), displaying contraceptive and HIV-entry inhibiting properties, and nucleoside reverse transcriptase inhibitors (NRTIs) (3'-azido-2',3'-dideoxythymidine (AZT), 3'-fluoro-2',3'-dideoxythymidine (FLT), or 2',3'-dideoxy-3'-thiacytidine (3TC)) were designed to simultaneously provide contraceptive and anti-HIV activity. Two linkers, acetate and succinate, were used to conjugate the nucleoside analogs with CS. The conjugates containing cellulose sulfate-acetate (CSA) (e.g., AZT-CSA and FLT-CSA) were found to be more potent than CS and other conjugates (e.g., AZT-succinate-CS, and FLT-succinate-CS). The presence of both sulfate and the acetate groups on cellulose were critical for generating maximum anti-HIV activity. In addition to showing equal potency against wild-type and multidrug resistant HIV-1, the AZT-CSA conjugate displayed significant contraceptive activity in an animal model, providing the initial proof-of-concept for the design and synthesis of dual-activity compounds based on these combinations.

PEGylation and Cell-Penetrating Peptides: Glimpse into the Past and Prospects in the Future.

Several drug molecules have shown low bioavailability and pharmacokinetic profile due to metabolism by enzymes, excretion by the renal system, or due to other physiochemical properties of drug molecules. These problems have resulted in the loss of efficacy and the gain of side effects associated with drug molecules. PEGylation is one of the strategies to overcome these pharmacokinetic issues and has been successful in the clinic. Cell-penetrating Peptides (CPPs) help to deliver molecules across biological membranes and could be used to deliver cargo selectively to the intracellular site or to the drug target. Hence CPPs could be used to improve the efficacy and selectivity of the drug. However, due to the peptidic nature of CPPs, they have a low pharmacokinetic profile. Using PEGylation and CPPs together as a component of a drug delivery system, the and efficacy of drug molecules could be improved. The other important pharmacokinetic properties such as short half-life, solubility, stability, absorption, metabolism, and elimination could be also improved. Here in this review, we summarized PEGylated CPPs or PEGylation based formulations for CPPs used in a drug delivery system for several biomedical applications until August 2019.

Application of solid-phase chemistry for the synthesis of 3'-fluoro-3'-deoxythymidine.

Reported solution-phase methods for the synthesis of 3'-fluoro-3'-deoxythymidine (FLT) are cumbersome, require purification of intermediates, and include several protecting/deprotecting steps. To circumvent these problems, a solid-phase strategy was designed for the synthesis of FLT. Thymidine was immobilized on trityl resin via the 5'-hydroxyl group. The subsequent mesylation of the free 3'-hydroxyl group in the presence of methanesulfonyl chloride afforded the polymer-bound 3'-O-mesylthymidine. Nucleophilic substitution of the mesyl moiety by hydroxyl group in the presence of sodium hydroxide produced the polymer-bound threothymidine. Fluorination with diethylaminosulfur trifluoride followed by acidic cleavage of the polymer-bound FLT in the presence of trifluoroacetic acid afforded FLT.

Selective diphosphorylation, dithiodiphosphorylation, triphosphorylation, and trithiotriphosphorylation of unprotected carbohydrates and nucleosides.

[chemical reaction: see text]. Aminomethyl polystyrene resin-bound linkers of p-acetoxybenzyl alcohol were subjected to reactions with diphosphitylating and triphosphitylating reagents to yield the corresponding polymer-bound diphosphitylating and triphosphitylating reagents, respectively. A number of unprotected carbohydrates and nucleosides were reacted with the polymer-bound reagents. Oxidation with tert-butyl hydroperoxide or sulfurization with Beaucage's reagent, followed by removal of cyanoethoxy group with DBU and the acidic cleavage, respectively, afforded only one type of monosubstituted nucleoside and carbohydrate diphosphates, dithiodiphosphates, triphosphates, and trithiotriphosphates with high regioselectivity.

Polymer-bound oxathiaphospholane: a solid-phase reagent for regioselective monothiophosphorylation and monophosphorylation of unprotected nucleosides and carbohydrates.

Two polymers bound to N,N-diisopropylamino-1,3,2-oxathiaphospholane were reacted with unprotected carbohydrates and nucleosides in the presence of 1H-tetrazole, followed by oxidation with tert-butyl hydroperoxide or sulfurization with Beaucage's reagent. The 1,3,2-oxathiaphospholane ring-opening with 3-hydroxypropionitrile, followed by treatment with DBU, afforded the corresponding monophosphate and monothiophosphate derivatives, respectively, through the elimination of polymer-bound ethylene episulfide. Reactions using this strategy offer the advantages of high regioselectivity, monosubstitution, and facile isolation and recovery of products.

Bilayer disruption and liposome restructuring by a homologous series of small Arg-rich synthetic peptides.

The effects of a series of low molecular weight water-soluble cationic linear peptide analogs (LPAs, <1000 MW) with increasing hydrophobic/hydrophilic balance on lipid bilayer phase behavior and permeability were examined using liposomes composed of zwitterionic dipalmitoylphosphatidylcholine (DPPC) and mixed zwitterionic/anionic DPPC/dipalmitoylphosphatidylglycerol (DPPG) lipid bilayers. LPAs were synthesized using a previously reported alkyl linkage strategy as Arg-C(n)-Arg-C(n)-Lys, where C(n) represents the saturated alkyl linkage separating the cationic residues (n=4, 7, or 11) (Ye et al., 2007 [1]). Differential scanning calorimetry results show that the cationic LPAs bound to and disrupted DPPC and, to a greater extent, DPPC/DPPG phase behavior. When added to preformed unilamellar liposomes, the LPAs led to significant structural changes based on cryogenic transmission electron microscopy (cryo-TEM). Coupling cryo-TEM with carboxyfluorescein leakage studies indicate that the LPAs induced permeabilization through bilayer expansion, which caused membrane thinning. The effects were inconsistent with increasing LPA hydrophobicity, which suggests that a cooperative effect between electrostatic binding and hydrophobic insertion determined the location of LPAs within the bilayer and their membrane activity. Our results for LPA-induced membrane disruption correlate with previous breast cancer cell uptake studies that showed minimal LPA-C(4) uptake, but high LPA-C(11) uptake through a non-endocytic mechanism.

Synthesis of nucleoside mono-, di-, and triphosphoramidates from solid-phase cyclosaligenyl phosphitylating reagents.

Chloromethyl polystyrene resin was reacted with 5-hydroxysalicylaldehyde in the presence of potassium carbonate to afford polymer-bound 2-hydroxybenzaldehyde. Subsequent reduction with borane solution produced polymer-bound 2-hydroxybenzyl alcohol. The reaction of immobilized 2-hydroxybenzyl alcohol with appropriate phosphitylating reagents yielded solid-phase cycloSaligenyl mono-, di-, and triphosphitylating reagents, which were reacted with unprotected nucleosides, followed by iodine oxidation, deprotection of cyanoethoxy groups, and the basic cleavage, respectively, to afford 5'-O-nucleoside mono-, di-, and triphosphoramidates in 52-73% overall yield.

Solid-supported diphosphitylating and triphosphitylating reagents for nucleoside modification.

This unit describes procedures for synthesis of diphosphitylating and triphosphitylating reagents. The synthesized reagents are first immobilized on appropriate polymer-bound linkers. Rigid and sterically hindered polymer-bound diphosphitylating and triphosphitylating reagents are then reacted selectively with the 5'-hydroxyl group of nucleosides in the presence of excess nucleosides. Typical oxidation with tert-butyl hydroperoxide, deprotection, and final cleavage of the products from the resins using a trifluoroacetic acid cocktail afford various nucleoside 5'-O-diphosphate and nucleoside 5'-O-triphosphate analogs. The use of the diphosphitylating and polymer-bound diphosphitylating reagents in preparation of oligodeoxynucleotides containing diphosphodiester internucleotide bridges is also described. This solid-phase strategy allows for the synthesis of the phosphorylated compounds without the need for nucleoside phosphate precursors, protected nucleosides, or purification of intermediates.

Application of a solid-phase beta-triphosphitylating reagent in the synthesis of nucleoside beta-triphosphates.

A beta-triphosphitylating reagent was subjected to reaction with aminomethyl polystyrene resin-bound p-acetoxybenzyl alcohol to yield the corresponding polymer-bound beta-triphosphitylating reagent. The solid-phase reagent was reacted with unprotected nucleosides (e.g., 3'-azido-3'-deoxythymidine, cytidine, thymidine, uridine, inosine, or adenosine) in the presence of 1H-tetrazole. Polymer-bound nucleosides underwent oxidation with t-butyl hydroperoxide, deprotection of cyanoethoxy groups with DBU, and the acidic cleavage, respectively, to afford only monosubstituted 5'-O-beta-triphosphorylated nucleosides.

Synthesis of polymer-bound 4-acetoxy-3-phenylbenzaldehyde derivatives: applications in solid-phase organic synthesis.

Aminomethyl polystyrene resin was reacted with 4-(5'-formyl-2'-hydroxyphenyl)benzoic acid and 4-(5'-formyl-2'-hydroxyphenyl)phenyl propionic acid, respectively, in the presence of 1-hydroxybenzotriazole and 1,3-diisopropylcarbodiimide to yield polymer-bound benzaldehydes. The phenolic group in resins was acetylated with acetic anhydride to afford two polymer-bound 4-acetoxybenzaldehydes. The reductive amination of polymer-bound linkers by amines and sodium triacetoxyborohydride, followed by sulfonylation with arylsulfonyl chloride derivatives in the presence of pyridine and the cleavage with TFA/DCM/H2O, produced pure sulfonamides.

Polymer-supported reagents for methylphosphorylation and phosphorylation of carbohydrates.

Two polymer-supported reagents for methylphosphorylation and phosphorylation of carbohydrates have been developed. p-Hydroxybenzyl alcohol and beta-mercaptoethanol were immobilized on cross-linked divinylbenzene-polystyrene copolymer and conjugated with methyl N,N-diisopropylchlorophosphoramidite. Carbohydrates were reacted with polymer-bound phosphitylating reagents. Further oxidation, with or without the methoxy group deprotection, and cleavage yielded methylphosphorylated or phosphorylated carbohydrates, respectively.