Hyaluronic Acid Conjugates as Vectors for the Active Targeting of Drugs, Genes and Nanocomposites in Cancer Treatment

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Hyaluronic acid is a high molecular weight (106 –107 Da) glycosaminoglycan polymer composed of repeating disaccharides: ?1,3 N-acetyl glucosaminyl-? 1,4 glucuronide. HA is ubiquitous, being the main component of extracellular matrix, and is essential for proper cell growth, structural stability of organs, and tissue organization. From the pharmaceutical standpoint, HA is a promising component, because it is biodegradable, biocompatible, nontoxic, hydrophilic, and nonimmunogenic. HA contains several chemical groups to which other components can be conjugated. The carboxylate on the glucuronic acid, the N-acetylglucosamine hydroxyl, and the reducing termination, have all been successfully utilized in conjugation reactions with drugs. The acetyl group may be enzymatically removed from the N-acetylglucosamine, and is thus also a potential site for drug conjugation. Low levels of the hyaluronic acid receptor CD44 are found on the surface of epithelial, hematopoietic, and neuronal cells; it is overexpressed in many cancer cells, and in particular in tumor-initiating cells. HA has recently attracted considerable interest in the field of developing drug delivery systems, having been used, as such or encapsulated in different types of nanoassembly, as ligand to prepare nano-platforms for actively targeting drugs, genes, and diagnostic agents. This review describes recent progress made with the several chemical strategies adopted to synthesize conjugates and prepare novel delivery systems with improved behaviors.
Cancer is a leading cause of death worldwide, accounting for 7.6 million deaths (around 13% of all deaths) in 2008. Deaths from cancer worldwide are projected to continue rising, with an estimated 13.1 million deaths in 2030. Lung, stomach, liver, colon and breast cancers cause the most cancer deaths each year.Conjugation of cytotoxic drugs with macromolecules improves their pharmacokinetic profile, prolonging the distribution and elimination phases. Furthermore, the slow release of active drug from the carrier may result in sustained high intratumoral drug levels and lower plasma concentrations of the active drug. In order to achieve this combined effect, a macromolecule-drug conjugate should preferentially release the active drug within the tumor tissue. The following components are essential to reach this goal: a biodegradable linkage, a suitable spacer, and a potent bioactive anticancer agent. Among the most widely studied macromolecules are N-(2-hydroxypropyl) methacrylamide (HPMA), polyglutamate, human serum albumin, dextrans, heparin, chitosan, dendrimers, multi-arm polyethylene glycol (PEG), and hyaluronic acid.The molecular weight of native HA has a wide range. In water, high molecular weight HA self-aggregates to form a viscous solution in which each molecule forms a sponge-like matrix with a radius of about 100 nanometers; this makes it a suitable candidate for passive tumor accumulation. In addition, HA plays an important physiological role in the tumorigenesis process, and consequently HA receptors are overexpressed on many types of tumor cells. This feature could be exploited in drug delivery, by using the receptor as an anchor to attach prodrugs or nanomedicine-based delivery systems, through a ligand, so as to increase the efficiency of anticancer drugs. (Molecules 2014, 19(3), 3193-3230.)

Biomacromolecules as carriers in drug delivery and tissue engineering

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Natural biomacromolecules have attracted increased attention as carriers in biomedicine in recent years because of their inherent biochemical and biophysical properties including renewability, nontoxicity, biocompatibility, biodegradability, long blood circulation time and targeting ability. Recent advances in our understanding of the biological functions of natural-origin biomacromolecules and the progress in the study of biological drug carriers indicate that such carriers may have advantages over synthetic material-based carriers in terms of half-life, stability, safety and ease of manufacture. In this review, we give a brief introduction to the biochemical properties of the widely used biomacromolecule-based carriers such as albumin, lipoproteins and polysaccharides. Then examples from the clinic and in recent laboratory development are summarized. Finally the current challenges and future prospects of present biological carriers are discussed.
Natural-origin biomacromolecules perform a diverse set of functions in their native setting. For example, polysaccharides function in membranes, intracellular communication and as storage sites, whereas proteins function as structural materials, transport vehicles, nutrients and catalysts. Transport proteins as carriers for delivery of nutrients and other necessary molecules are of special interest. Inspired by such natural processes in organisms, scientists started to utilize natural and biological macromolecules, including proteins, polysaccharides, and lipoproteins, for the delivery of drugs and tissue engineering. Biomacromolecule-based drug carriers are nontoxic, non-immunogenic and have high drug loading content, good biocompatibility and targeting ability. Meanwhile, they are also capable of controlled and sustained drug release. Many meaningful designs have been reported using biological carriers, some of which are already approved for clinical use. Biomacromolecules used as carriers include proteins (albumin, transferrin, lipoproteins, silk fibroin, collagen, keratin) and polysaccharides (chitosan, cyclodextrin, hyaluronic acid, heparin and pectin). The structural diagrams of these carriers are shown in Fig. 1. These biomacromolecules can be naturally obtained from animals and plants in abundant amounts and are renewable resources. They have good affinity to organisms and weak immune rejection, and can be degraded by in vivo enzymes; the metabolites also have low toxicity to organisms. Biomacromolecule-based carriers have been reported in the form of prodrugs, drug conjugates, nanoparticles, microcapsules, hydrogels and tissue engineering scaffolds. The use of biomacromolecule-based carriers has been shown to improve the pharmacokinetics of the payloads and to reduce systemic toxicity and immunogenicity. Furthermore, the hydroxyl, amine and carboxyl groups on the chains of these biomacromolecules can be utilized for chemical modification, making them of great significance in biomedical field.(Acta Pharm Sin B. 2018 Jan; 8(1): 34–50.)

Novel Spermine-Derivative Cationic Lipids as an Effective Gene Carrier In Vitro

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In the present study, nonionic surfactant vesicles (niosomes) formulated with Span 20, cholesterol, and novel synthesized spermine-based cationic lipids with four hydrocarbon tails in a molar ratio of 2.5:2.5:1 were investigated as a gene carrier. The effects of the structure of the cationic lipids, such as differences in the acyl chain length (C14, C16, and C18) of the hydrophobic tails, as well as the weight ratio of niosomes to DNA on transfection efficiency and cell viability were evaluated in a human cervical carcinoma cell line (HeLa cells) using pDNA encoding green fluorescent protein (pEGFP-C2). The niosomes were characterized both in terms of morphology and of size and charge measurement. The formation of complexes between niosomes and DNA was verified with a gel retardation assay. The transfection efficiency of these cationic niosomes was in the following order: spermine-C18 > spermine-C16 > spermine-C14. The highest transfection efficiency was obtained for transfection with spermine-C18 niosomes at a weight ratio of 10. Additionally, no serum effect on transfection efficiency was observed. The results from a cytotoxicity and hemolytic study showed that the cationic niosomes were safe in vitro. In addition, the cationic niosomes showed good physical stability for at least 1 month at 4°C. Therefore, the cationic niosomes offer an excellent prospect as an alternative gene carrier.(Gene therapy has been widely endorsed as a promising therapeutic approach for many incurable diseases related to gene function, such as genetic diseases, cancer, cardiovascular diseases, and autoimmune diseases. Successful gene therapy requires not only therapeutically suitable genes but also a safe and efficient gene carrier. To avoid severe side effects resulting from viral vectors, such as immunogenicity, mutagenesis, and carcinogenesis, nonviral vectors offer an attractive alternative. Cationic liposomes, a vesicular system widely investigated as effective gene carriers, are one of the preferred nonviral vectors. In addition, several studies have reported on the use of another vesicular system, nonionic surfactant vesicles (niosomes), as a gene carrier that can potentially be substituted for liposomes.Niosomes are nonionic surfactant vesicles formed by the self-assembly of nonionic amphiphiles into a bilayer structure in an aqueous medium. The nonionic surfactants preferably used to prepare niosomes include alkyl ethers and alkyl glyceryl ethers (Brij), sorbitan fatty acid esters (Span), and polyoxyethylenefatty acid esters (Tween). However, niosomes have several advantages over liposomes, including low production costs, high purity, uniform content, greater stability, and the ease of storing nonionic surfactants. Cationic niosomes used as gene carriers are usually composed of nonionic surfactants (i.e., Tween and Span), cholesterol, and cationic lipids. One of the major factors affecting gene transfection mediated by cationic niosomes is niosome composition, including the types of surfactants and cationic lipids used.
The cationic lipids used as transfection reagents usually contain three parts: a hydrophobic group, a linker group, and a positively charged head group that can interact with DNA and cause DNA condensation. The polyamines furnish one of the most effective cationic lipid head groups. Among the polyamines, spermine, a well-known polyamine consisting of a tetraamine with two primary and two secondary amino groups, plays an important role as a gene carrier. Spermine-derivative cationic lipids commercially available for gene delivery applications include dioctadecylamidoglycylspermine (DOGS) and dipalmitoylphosphatidyl ethanolamidospermine (DPPES). In the present study, cationic niosomes formulated with Span, cholesterol, and novel synthesized spermine-based cationic lipids with four hydrocarbon tails, namely, Tetra-(N1,N1,N14,N14-myristeroyloxyethyl)-spermine (spermine-C14), Tetra-(N1,N1,N14,N14-palmitoyloxyethyl)-spermine (spermine-C16), and Tetra-(N1,N1,N14,N14-steroyloxyethyl)-spermine (spermine-C18), in a molar ratio of 2.5:2.5:1 were investigated as gene carriers. Factors affecting transfection efficiency and cell viability, including cationic lipid structure (i.e., differences in the acyl chain length (C14, C16, and C18) of the hydrophobic tails as well as the weight ratio of niosomes to DNA) were evaluated in a human cervical carcinoma cell line (HeLa cells) using pDNA-encoded green fluorescent protein (pEGFP-C2). The morphology, size, and charge of these niosomes/DNA complexes were characterized, and agarose gel electrophoresis was performed. Moreover, the physical stability of these cationic niosomes was evaluated with size and charge measurements.) (AAPS PharmSciTech. 2014 Jun; 15(3): 722–730.)

Reduction Sensitive Lipid Conjugates of Tenofovir: Synthesis, Stability, and Antiviral Activity

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The therapeutic value of numerous small molecules hinges on their ability to permeate the plasma membrane. This is particularly true for tenofovir (TFV), adefovir, and other antiviral nucleosides that demonstrate potent antiviral activity but poor bioavailability. Using TFV as a model substrate, we hybridized two disparate prodrug strategies to afford novel reduction-sensitive lipid conjugates of TFV that exhibit subnanomolar activity toward HIV-1 and are stable in human plasma for more than 24 h with a therapeutic index approaching 30000. These compounds significantly rival the clinically approved formulation of TFV and revitalize the potential of disulfide-bearing prodrugs which have seen limited in vitro and in vivo success since their debut over 20 years ago. We further demonstrate the utility of these conjugates as a tool to indirectly probe the enzymatic hydrolysis of phosphonomonoesters that may further advance the development of other prodrug strategies for nucleosides, peptides, and beyond.
We sought to design disulfide-linked lipid conjugates that readily permeate the plasma membrane and rapidly releases the nucleoside within the target cell without the need for enzymatic activation. In contrast to other prodrug strategies, our design relies on the reduction-sensitive nature of the S–S bond that selectively bows to the universal glutathione redox potential that exists at the plasma membrane between the extra- and intracellular milieu.Glutathione, often referred to as GSH, is a tripeptide typically stockpiled in millimolar concentrations (2–10 mM) within the cytosol that participates in a multitude of biological functions to promote the reduction of disulfides, detoxification of xenobiotics, mediate immunoregulation, and curb oxidative stress32 among others. GSH is seldom found in the extracellular space and thus creates a gradient that fosters intracellular reduction of disulfides and simultaneously promotes an oxidative environment to support S–S formation in the surrounding media. (J Med Chem. 2016 Aug 11; 59(15): 7097–7110.)

Polyamine-based analogs and conjugates as antikinetoplastid agents

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Naturally occurring polyamines: putrescine, spermidine and spermine are crucial for Kinetoplastid growth and persistence. These aliphatic polycations are either biosynthesized or internalized into Kinetoplastid by active transport. Impairing the polyamine metabolism using polyamine derivatives is an interesting path in the search of new antikinetoplastid chemotherapy. In the past 30 years, research interest in this field has been constantly expanding and recent results demonstrated that the discovery of a polyamine-based antikinetoplastid drug is undoubtedly possible. In this paper, all the polyamine derivatives previously described to present an antikinetoplastid activity are reported. This review is organized around three main parts which are diamine, triamine and tetramine derivatives. Each part includes the description of the series of molecules and, their in vitro and in vivo activity when available. Structure-activity relationships of these derivatives are discussed and the most promising structures for a positive outcome are eventually highlighted.(European Journal of Medicinal Chemistry.,Volume 139, 20 October 2017, Pages 982-1015.)

Synthesis of peptide-protein conjugates using N-succinimidyl carbamate chemistry

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Peptide-protein conjugates are useful tools in different fields of research as, for instance, the development of vaccines and drugs or for studying biological mechanisms, to cite only few applications. N-Succinimidyl carbamate (NSC) chemistry has been scarcely used in this area. We show that unprotected peptides, featuring one lysine residue within their sequences, can be converted in good yield into NSC derivatives by reaction with disuccinimidylcarbonate (DSC). No hydrolysis of the NSC group was observed during RP-HPLC purification, lyophilization, or storage. NSC peptides reacted efficiently within minutes with lysozyme used as model protein. To illustrate usefulness of the method consisting of the synthesis of a peptide-protein conjugate of biological interest, a NSC peptide derived from a peptide substrate for tyrosylprotein sulfotransferase (TS) was synthesized and ligated to receptor-binding nontoxic B-subunit of Shiga toxin (STxB). Immunofluorescence studies showed the intracellular delivery of the TS-STxB conjugate and its ability to circulate to the Golgi as the native STxB protein. Moreover, we demonstrate that the TS label could be sulfated by tyrosylprotein sulfotransferases present in the Golgi. Thus, NSC chemistry permitted rapid synthesis of a peptide-protein conjugate worthwhile for studying the transport of proteins from the plasma membrane to the Golgi. The second part of this article describes a more general method for synthesizing peptide-protein conjugates without any limitation of the peptide sequence. The conjugates were assembled by combining NSC chemistry and alpha-oxo semicarbazone ligation. To this end, a glyoxylyl NSC peptide was synthesized and reacted with lysozyme. The glyoxylyl groups on the protein were then reacted with a semicarbazide peptide to produce the target peptide-protein conjugate. Both reactions, namely, urea bond formation and alpha-oxo semicarbazone ligation, were carried at pH 8.0 using a one-pot procedure.(Bioconjug Chem. 2010 Feb 17;21(2):219-28. )

Synthesis of doxorubicin cell‐penetrating peptide bioconjugates

Cell‐penetrating peptides, also known as protein transduction domain (PTD), have attracted interest as carriers for intracellular drug delivery. We report the first drug conjugate with a negatively charged amphipathic cell‐penetrating peptide. Furthermore, we compare two different doxorubicin cell‐penetrating peptide conjugates, which are both unique in their properties, due to their net charge at physiological pH, namely the positively charged octaarginine and the negatively charged proline‐rich amphipathic peptide. These conjugates were prepared exploiting a novel heterobifunctional crosslinker to join the N‐terminal cysteine residue of the peptides with the aliphatic ketone of doxorubicin. This small linker contains an activated thiol as well as aminooxy functionality, capable of generating a stable oxime bond with the C‐13 carbonyl group of doxorubicin. The disulfide bond formed between the peptide and doxorubicin enables the release of the drug in the cytosol, as confirmed by drug‐release studies performed in the presence of glutathione. Additionally, the cytotoxicity as well as the cellular uptake and distribution of this tripartite drug delivery system was investigated in MCF‐7 and HT‐29 cell lines.

Heterobifunctional crosslinkers are extensively applied in drug modifications, and their utilization has proven to be very important for the attachment of diverse carriers, such as monoclonal antibodies, proteins, polymers, and peptides, to doxorubicin. Established conjugation technique is applied at the C‐13 keto group by hydrazones, due to their fast hydrolysis in acidic environment existing in biological compartments like endosomes and lysosomes. However, the insufficient stability of the doxorubicin hydrazone conjugates has been reported even at physiological pH (7.4), leading to the release of the free drug in the bloodstream. In order to overcome these difficulties, we have chosen a crosslinker capable of creating an oxime bond on doxorubicin’s ketone, due to the higher hydrolytic stability of the oxime group. Additionally, thiol‐containing carriers, like albumin proteins, have also been conjugated to anthracyclines utilizing functional groups that are highly specific for sulfhydryl groups, e.g. maleimides and pyridyl disulfides. The application of pyridyl disulfides is advantageous, because a disulfide bond between the linker and the cargo is formed, which can be reduced in the cytoplasm by glutathione to deliver the freight. Furthermore, pyridyl disulfides can serve as a protective group during synthesis to avoid undesired dimerization as well as an activating group for the thiol to facilitate disulfide formation. In contrast, maleimides react with a thiol via Michael addition; thus, a covalent bond is created that cannot be cleaved under physiological conditions. Therefore, we have selected a heterobifunctional crosslinker that contains a protected aminooxy group and pyridyl disulfide.

Unraveling the Anticancer Effect of Curcumin and Resveratrol

Resveratrol and curcumin are natural products with important therapeutic properties useful to treat several human diseases, including cancer. In the last years, the number of studies describing the effect of both polyphenols against cancer has increased; however, the mechanism of action in all of those cases is not completely comprehended. The unspecific effect and the ability to interfere in assays by both polyphenols make this challenge even more difficult. Herein, we analyzed the anticancer activity of resveratrol and curcumin reported in the literature in the last 11 years, in order to unravel the molecular mechanism of action of both compounds. Molecular targets and cellular pathways will be described. Furthermore, we also discussed the ability of these natural products act as chemopreventive and its use in association with other anticancer drugs.

Papers describing curcumin and/or resveratrol are present in almost fifteen thousand of publications in the last ten years. Both polyphenols have been described as promising anticancer compounds; however, the mode of action for them are still unclear and not fully comprehended.

Curcumin (diferuloylmethane) is an active ingredient of the perennial herb Curcuma longa, also known as turmeric. The yellow color of this polyphenol is chemically related to its major fraction, which contains curcuminoids. Curcumin has been used for a long time in countries such as China and India as traditional medicines. This ancient remedy has brought the attention of scientific community for a wide range of beneficial properties including anti-inflammatory, antioxidant and chemopreventive.

By the other hand, resveratrol (trans-3,5,4′-trihydroxystilbene) is a stilbene phytoalexin synthetized by a variety of plants, specially vine in response to fungi infections and ultraviolet radiation. This compound is found at high concentration in grapes and red wine, which antioxidant effect is well established in several different assays. Resveratrol has been investigated as potential compound for the treatment of several diseases, regulation of immune system and chemoprevention. (Nutrients. 2016 Nov; 8(11): 628. )