New developments and clinical transition of hyaluronic acid-based nanotherapeutics for treatment of cancer: reversing multidrug resistance, tumour-specific targetability and improved anticancer efficacy

Abstract

This review aims to overview and critically analyses recent developments in achieving tumour-specific delivery of anticancer agents, maximizing anticancer efficacy, and mitigating tumour progression and off-target effects. Stemming from critical needs to develop target-specific delivery vehicles in cancer therapy, various hyaluronic acid (HA)-conjugated nanomedicines have been fabricated owing to their biocompatibility, safety, tumour-specific targetability of drugs and genes, and proficient interaction with cluster-determinant-44 (CD44) receptors over-expressed on the surface of tumour cells. HA-based conjugation or surface modulation of anticancer drugs encapsulated nanocarriers have shown promising efficacy against the various types of carcinomas of liver, breast, colorectal, pancreatic, lung, skin, ovarian, cervical, head and neck and gastric. The success of this emerging platform is assessed in achieving the rapid internalization of anticancer payloads into the tumour cells, impeding cancer cells division and proliferation, induction of cancer-specific apoptosis and prevention of metastasis (tumour progression). This review extends detailed insight into the engineering of HA-based nanomedicines, characterization, utilization for the diagnosis or treatment of CD44 over-expressing cancer subtypes and emphasizing the transition of nanomedicines to clinical cancer therapy.

Keywords:

• Cancer
• CD44 overexpression
• hyaluronic acid
• nanomedicines
• tumour-specific targetability
• improved anticancer efficacy

Introduction

Cancer is a group of diseases involving an abnormal growth of cells which tend to proliferate in an uncontrolled way and, in some cases, to metastasize to surrounding tissues. It tends to remain the major cause of fatalities world-wide with an expected forecast of a staggering 1.68 million new diagnostic cases and 0.6 million deaths for the year 2017 in the USA alone. Though the statistical death rate from cancer (from 2005 onwards) has experienced a decline by 1.5% annually, it is still a far-cry from the desired cancer survival rates [1]. The transformation from normal cells to cells that can form a clear mass to outright cancer involves multifarious steps, process known as malignant progression. This results from the interaction between genetic factors and detrimental external agents; mainly physical, chemical and biological carcinogens.

For decades, scientists have been attempting to find promising cures for the said menace. Current cancer clinical therapies include chemotherapy, surgery and radiotherapy. However, certain limitations associated with these therapeutic regimes limits their prolific use; namely undesired cytotoxic effects led by non-specific drug accumulation, and reduced efficacy on account of poor solubility associated with chemotherapeutics. Moreover, other adverse effects including cardiotoxicity and myelosuppression occurred in chemotherapy; alongside infection, bleeding, or irritation in radiotherapy and surgery respectively, are also commonly employed [2]. Therefore, novel drug delivery strategies have been explored to eradicate unspecified issues associated with conventional cancer therapies, to improve tumour-specific targetability, to alleviate tumour progression and improve anticancer efficacy.

The use of nanotechnology-based targeted therapies has gained remarkable attention of the researchers in recent years. These nanotechnology-based approaches have been rigorously investigated for their role in cancer imaging and as biocompatible carriers in the proficient delivery of chemotherapeutic agents, elevating blood circulation time, and producing optimal therapeutic effects by decreasing the residual toxicity and non-specific drug accumulation [3].

Mechanistically, nanocarriers may be directed to the tumorigenic site by one of two processes: passive targeting or active targeting. In the former process, the enhanced permeability and retention effect aids nanomedicines to be passively extravagated through leaky vasculature, resulting in accumulation at the tumour site. However, this may lead to non-specific drug diffusion which may expose normal cells to cytotoxic effects of anticancer agents. Active targeting has thus been stepped up as a promising solution whereby the therapeutic efficacy of drugs is enhanced by the increased accumulation and cellular uptake through receptor-mediated endocytosis (RME). Nanocarriers may be synthesized to accommodate ligands, thus enhancing their ability to interact with endothelial cell surface receptors which are over-expressed on the surfaces of cancer cells. Hyaluronic acid (HA) cellular uptake receptors, CD44 and RHAMM, are such examples of over-expressed receptors on the surface of cancer cells, and thus have been exploited for the generation of target-specific drug delivery of these nanocarriers [4].

Hyaluronic acid (HA)

Hyaluronic acid (HA), a natural mucopolysaccharide uniting from N-acetyl-D-glucosamine and D-glucuronic acid chain, occurs in vast majorities of animal species and is found to be a vital element of extracellular matrix. It is synthesized by the action of hyaluronan synthases at the inner surface of the plasma membrane and undergoes degradation by hyaluronidase (HAase)-mediated enzymatic hydrolysis of its β-1,4-glycosidic bonds. Aside from demonstrating non-allergenic, non-toxic and biocompatible properties, a multitude of biological functions have also been attributed to HA including wound healing [4], tissue regeneration, anti-wrinkle effects, anti-inflammatory properties and cancer prognosis [5]. Additionally, HA has been proved to play pivotal roles in modulating various biological activities, such as (i) signal transduction, (ii) cell motility, (iii) embryogenesis, (iv) angiogenesis, (v) granulation tissue formation and repair, and (vi) cancer invasiveness and metastasis [6].

Hyaluronic acid in drug design, development and drug delivery

HA has contributed pivotal roles in drug delivery due to its versatile properties including biodegradability, biocompatibility, several opportunities for chemical modifications and inherent targeting properties. The earliest HA-drug conjugate was designed to produce prodrugs that would demonstrate stability whilst in blood circulation and cleaved at the desired target site. To exploit the use in drug designing, a series of chemical modifications can be carried out on the three distinct functional regions of HA, namely, the acetamido, hydroxyl and carboxylic groups.

The most widely studied HA-drug conjugates comprise of anticancer agents, as HA receptors RHAMM and CD44 show over-expression in multiple cancer types [7]. Thus, HA-drug conjugates offer several advantages with regards to drug solubility, stability, targeting and controlled release properties. Once taken up by the cells, intracellular enzymes lead to hydrolysis of the HA-drug bond, thereby causing release of entrapped drugs inside the targeted cancer cell [8]. Till date, numerous HA-conjugated delivery systems have been attempted for the tumour-specific delivery of anticancer moieties, for the production of novel anticancer therapeutics with improved antitumor efficacy.

Anticancer potential of HA: Mechanistic insight and CD44 receptors

Over-expressed receptors on the surface of cancerous cells, such as folate, transferrin and epidermal growth factors constitute the basis of selectivity amongst malignant and normal cells. CD44 is one of the most important surface receptor holding great interest of scientific community for years. Specifically, CD44 (also referred to as H-CAM) is a stem-like cellular receptor (transmembrane glycoprotein) associated with cancer progression, metastasis, lymph node infiltration and development of malignancies. CD44 receptors also serve as main biomarker in the detection of various cancer cell types, such as hepatocellular carcinoma, basal-type breast cancer and ovarian cancer [9,10]. In addition, CD44 and its associated variants assume important roles as early detection tools for the prediction of cancer recurrence. For example, CD44v9, a gastric cancer marker, may also serve to detect cancer’s reversion in earlier treated patients [11].

HA and CD44 interaction stimulates a series of signalling pathways leading to adhesion, metastasis and progression of cancer cells. For example, RhoA, Cdc42, ankyrin, metalloproteinase-9 (MMP-9) and tyrosine kinase receptor activation, and associated downstream signalling, is initiated by direct interaction between CD44 and HA. Likewise, Wnt/β-catenin pathway is triggered upon interaction of HA with a variant CD44v6, resulting in exhibition of metastasis in colon cancer stem cells [12]. Studies involving role of HA-CD44 binding in lung cancer development and progression demonstrated enhanced cellular proliferation via MAPK pathway [12]. Interestingly, significant inhibition in the Kras-stimulated cell proliferation was observed in lung adenocarcinoma in vivo, following gene-deletion responsible for encoding CD44 [13].

Despite of directly contributing promising role in cancer progression, HA-CD44 interaction has also been shown to play a pivotal role in ameliorating the emerging chemo-resistance against anticancer drugs. Mechanistically, the interaction tends to result in the ankyrin binding to MDR-1, causing the efflux of the oncotherapeutic resulting in chemo-resistance in cancer cells [14]. Likewise, chemo-resistance has also been demonstrated in triple negative breast cancer through HA-CD44 mediated activation of c-Jun signalling pathway and consequent upregulation of anti-apoptotic Bcl-2/IAP [15].

Hyaluronic acid-based nanomedicines

Ability of HA to selectively interact with cancer receptors (CD44 receptors) has gained immense interest of scientists, researchers and health professionals especially attempting in designing novel chemotherapeutic drugs. The innate characteristics of HA including non-immunogenicity, hydrophilic nature and bio-compatibility has gained phenomenal recognition in the development of nanomedicines that can aggressively target the abnormal genes and anticancer agents against cancer cells and diminish off-target adverse events. Hydrophilicity of HA results in increased solubility in aqueous media allowing water-based reaction mechanisms, hence pushing aside the use of potentially harmful non-polar organic solvents. Due to the hydrophobic chemical nature of various anti-cancer drugs, loading onto hydrophilic HA-based nanocarrier and within HA-based nanomedicines can potentially enhance their pharmacological efficacy [16]. Drug release and efficiency ratio depends majorly on the tight packing during absorption and non-target tissue travel and sustained release at the site of action. This is varied by pH changes, redox reactions and various other pharmacodynamic conditions. Researchers have shown HA-based formulations to neutralize these barriers and increase efficiency of their actions [17]. HA-based anticancer nanomedicines have not only enhanced anticancer efficacy against various types of cancers, but also significantly reduced their side effects [17]. Among other promising features of using HA as targeting ligand, its specific absorption and adsorption on CD44 receptors attributes the development HA-based nanocarriers [18].

Exploiting the said mechanisms, various HA-conjugated nanomedicines have been designed for efficient tumour-specific delivery of chemotherapeutic agents by specific interaction with CD44 receptors over-expressed on various tumours, such as liver, breast, colorectal, pancreatic, cervical, lung, gastric, ovarian, skin, head and neck cancers. These nanomdicines are engineered by fabricating several nanocarriers, such as polymeric nanoparticles (NPs) [19], metal nanocomposites [20], inorganic NPs, liposomes, micelles, solid lipid nanoparticles (SLNs), nanostructured lipid carriers (NLCs), lipoplexes, nanohydrogels, nanogels, nanomaterials [21], nanocomposites, nanoconjugates, carbon nanotubes [22] and nanostars [23].

Treatment of hepatocellular carcinoma: HA-conjugated or -decorated nanomedicines

In USA, the average rate of liver cancer continues to increase in men and women at 4 and 5%, respectively. From 2010 to 2014, a 3% rise in fatalities associated with liver cancer has been reported [1]. Due to continuous escalation of cancer associated fatalities, scientists and researchers are continuously attempting to formulate novel strategies for the treatment of the said menace. In an attempt to achieve tumour-specific delivery of pharmacological moieties to cancerous tissues and protein targets, several types of nanocarrier systems have been employed, such as supramolecular NPs [18], lipid hybrid NPs [24], metal NPs [25], polymeric NPs [26], micelles [27], nanogels [28] and multifunctional liposomes [29].

In an attempt to achieve simultaneous delivery of chemotherapeutic agent and gene, core-shell morphology-based supramolecular NPs were developed comprising of β-cyclodextrin-linked poly-l-lysine (PLCD) and were targeted against hepatocellular carcinoma (HCC) [18]. The surface of PLCD NPs was further modified with HA as targeting ligand against the CD44 over-expressing MHCC-97H cancer cells. The results from CCK-8 assay revealed that these HA-decorated NPs, bearing an average diameter of 195.8 nm, efficiently delivered doxycycline (DOX) and the gene inside the HCC cells via CD44, and RME and significantly prevented the cancer cells proliferation. In tumour xenograft MHCC-97H bearing mice, these NPs were rapidly accumulated within the cancerous tissues, illuminating efficient hepatoma-targetability, thereby exhibiting positive potential for treatment of HCC [18].

Another study was carried out in which sorafenib (SF)-conjugated HA-based lipid hybrid NPs (SF-HA-cLNS) were synthesized for targeted delivery against H22 hepatocarcinoma bearing xenograft murine model [24]. In this study, SF-HA-cLNS were prepared via nanoprecipitation followed by electrostatic absorption of HA on the surface, bearing an average diameter of 130 nm. Results from MTT assay showed that the final NPs conjugates were not only biocompatible, but also demonstrated concentration-dependent cytotoxicity against HepG2 cells which was significantly greater in contrast to free SF. The in vivo results showed that aside from efficient accumulation at the tumour site achieved through HA-mediated active targeting, SF-HA-cLNS elicited significant apoptosis in tumour cells [24].

For the target specific delivery of doxorubicin (DOX), functionalized NPs comprising of HA-glycyrrhetinic acid (HA-Cyst-GA) have also been fabricated and targeted against HCC [26]. These DOX-loaded HA-Cyst-GA NPs exhibited higher cytotoxicity and lower value of IC₅₀ against CD44 positive HepG2 cells in contrast to the free DOX. The tumour-specific targetability of DOX-loaded HA-Cyst-GA NPs was evaluated using non-invasive NIR imaging technique. Images from NIR imaging carried out on hepatocarcinoma H22 tumour allograft carrying mice highlighted selective penetration of DiR dye labelled NPs inside the tumorous mass in liver. Relevance to in vivo antitumor activity carried out on tumour HepG2 xenograft model, profound decrease in tumour mass was observed upon systemic treatment with DOX-loaded HA conjugated functionalized NPs in comparison to the free DOX [26].

Another nanodelivery system, micelles, was engineered by Xu et al. [27] comprising of quercetin (QU) and HA for improved in vivo docetaxel (DOC) therapy against HCC via HA-CD44 based targeting and QU-mediated p-gp inhibition. DOC-encapsulated HA-QU micelles (DTX/HA–QU) showed higher cellular accumulation via CD44-HA-based endocytosis, as well as effectively reached the tumour site via enhanced permeability and retention (EPR) factor. The in vitro cellular cytotoxicity studies revealed that DOC-HA-QU NPs extended 13.6-times greater activity in contrast to Taxotere^®, elucidated via IC₅₀ values. DTX-HA-QU NPs also exhibited most significant antitumor activity in a xenograft tumour-bearing mice model with 73.9% tumour inhibition ratio [27]. Additionally, the conjugated micelles effectively down-regulated the p-gp efflux pumps expression in cancer cells, suggesting their role as promising candidates for HC therapy [27]. These results indicated significant cytotoxicity in HCC, rapid uptake of anticancer drugs by CD44 positive HepG2 cells, profound internalization and enhanced anticancer activity against hepatic carcinoma bearing mice.

In order to optimize drug uptake into the cancer cells, a conjugated nanogel system was prepared employing methacrylation strategic route [28]. Cellular uptake studies, such as NIR imaging, penetration and biodistribution analysis highlighted that HA nanogels were rapidly accumulated inside HepG2 multicellular spheroids, owing to the receptor mediated endocytosis. In vivo studies illuminated that DOX-encapsulated HA nanogels demonstrated significantly greater antitumor efficacy in CD44 positive H22 hepatic tumour bearing mice in contrast to the free DOX [28].

By utilizing HA as targeting ligand, multifunctional liposomes (HA-R6H4-L) comprising of arginine–histidine rich R6H4 (a cell penetrating peptide) were also engineered for selective HepG2 cancer targeted delivery of paclitaxel (PTX) [29]. In this study, aside from its use as a targeting ligand, HA was also used for shielding the positive charge in R6H4-L for assembling HA-based R6H4-liposomes. These PTX-conjugated HA-R6H4-liposomes extended remarkable cytotoxicity against HepG2 hepatic carcinoma, significantly improved accumulation of PTX inside the tumour cells, and displayed remarkably higher anticancer activity against tumour xenograft murine model bearing hepatic carcinoma [29].

Treatment of breast cancer: HA-conjugated multifunctional nanomedicines

Breast cancer tends to be the second most prevalent cancer amongst women worldwide with an estimated death rate forecast of a startling 40,610 in USA alone for the year 2017 [1]. From amongst its various sub-types, triple-negative breast cancer (TNBC) poses serious concerns as its response level against the conventional chemotherapeutic regimens is substantially low. Additionally, significantly over-expressed CD44 receptors form the hallmark of various breast cancer subtypes, including TNBC. To achieve tumour-specific targetability, reduced drug resistance and improve anticancer efficacy, several HA-conjugated or -decorated nanocarrier systems, such as nanoassemblies [30], SLNs [31], NPs [32], graphene oxide (GO) NPs [33], poly-lactide-co-glycolide (PLGA)-NPs [34], micelles [35,36] and NLCs [37] have been fabricated for treatment of breast cancer.

Recently, Jeong et al. [30] prepared novel aminomethyl-boronic acid (APBA) conjugated HA ceramide (HACE) based nanoassemblies for targeted delivery of the natural anticancer compound, Manassantin B, against CD44 over-expressed MDA-MB 231 breast cancer cells. Manassantin B is a lignin extracted from the roots of Saurusus chinensis. The final nanoassemblies formed through amide linkage between APBA and HA exhibited enhanced accumulation of HA-APBA-MB nanoassemblies inside the tumour cells compared to the free MB which was attributed to the CD44-RME, in addition to sialic acid-pheylboronic acid interaction. These results clearly revealed proficient ability of HA as a tumour-targeting agent [30].

P-glycoproteins (p-gp) tend to play crucial roles in developing multidrug resistance (MDR) against chemotherapeutic agents by effluxing out anticancer drugs from the cancer cells. Exploiting the use of pluronic 85 (P-85) acting as an inhibitor of p-glycoprotein (p-gp), and HA as a targeting ligand, HA-P85 based SNLs were fabricated carrying PTX as the anti-neoplastic agent [31]. The anticancer efficacy of these SLNs (HA-P85-SNL) was tested against MCF-7 and breast cancer bearing Balb/c mice xenograft models. Besides showing optimal physicochemical characteristics, these nanocarriers showed 5.5 folds increase in bioavailability and significantly higher anticancer efficacy against breast carcinoma compared to the control groups [31].

CTGF (connective tissue growth factor), a physiologic factor responsible for the development of MDR, is pathologically over-expressed in breast cancers. Mechanistically, the up-regulation of CTGF tends to stimulate the cellular resistance to apoptosis protein 1 (cIAP1) and Bcl-xL, which are the true factors promoting drug resistance. Consequently, for the inhibition of cIAP1 and Bcl-xL, small interference RNA (siRNA) holds great promise for down-regulation of CTGF and consequent prevention of the associated drug resistance. In an attempt to alleviate the development of MDR, improving target-specific delivery of anticancer agents, and enhancing anticancer efficacy, mesoporous silica NPs were constructed coupled with breast cancer navigating and cell penetrating peptide (PEGA-pVEC) and HA [32]. These NPs were simultaneously loaded with siRNA and DOX and targeted against MBA-MD 231 cells. Results indicated that upon successfully penetrating the target cancer cells via dual activity of the penetrating peptide and HA, siRNA was able to silence the anti-apoptotic proteins further allowing DOX to execute its anticancer effect [32].

Oncogene directed therapy has gained remarkable attention of researchers for cancer imaging and anticancer therapy. HA based graphene oxide (GO) NPs were prepared for the delivery of Cy-3 florescence labelled antisense miR-21 nucleic acid, for the inhibition of oncogenic miR-21 in CD44 + MBA-MB231 cells [33]. The results established that the inhibition of miR-21 with the aid of HGP21 lead to reduced migration and proliferation of cancer cells alongside enhanced apoptosis, as observed via modulated caspase 3/7 activity.

For overcoming MDR, enhancing cellular uptake of anticancer agents and achieving target-specific internalization, HA-conjugated PLGA-NPs [34], unmodified micelles [35], peptide-modified micelles [36], NLCs [37] and multifunctional liposomes [38] have been engineered for targeted delivery of anticancer drugs against MDA-MB 231 breast cancer cells. Improved biocompatibility, reduced systematic toxicity, enhanced cellular internalization and apoptosis in cancer cells, inhibited angiogenesis and metastasis in cancerous cells and improved anticancer efficacy against MDR breast cancer have been evidenced [34–36].

To exploit the phenomenon of synergistic anticancer effects and simultaneous delivery of multimodal anticancer agents has also been attempted by engineering HA-conjugated NLCs [37]. An improved cellular uptake of anticancer drugs, enhanced cytotoxicity against of cancer cells, synergistic anticancer efficacy, reduced tumour volume and improved therapeutic outcomes have been observed [37].

Gamma (γ)-glutamylcyclotransferase (GGCT), an oncogenic protein translated from the open reading frame (ORF) 24 on chromosome 7, tends to be over-expressed in breast cancer, inducing uncontrolled cellular proliferation. To downregulate the protein, Ran et al. [38] adopted an RNA inference approach in which they constructed anti-GGCT siRNA-encapsulated HA-PEG-modified liposomes and tested against MDR CD44 over-expressing MCF-7 breast cancer. The conjugated HA-modified liposomes, bearing an average diameter of 216 nm, were able to effectively down-regulate GGCT at a dose of 0.35 mg/kg, consequently inducing cellular apoptosis in MCF-7/ADR cells whilst showing insignificant cytotoxicity against normal cells [38].

Micro RNAs (miRNA) play pivotal role in the modulation of pathways responsible for cancer resistance. One such miRNA, MiR-542–3p activates the tumour suppressor p53 while inhibiting survivin (anti-apoptotic molecule). Wang et al. [39] thus focused on developing HA-based PEI-PLGA NPs for the targeted delivery of MiR-542–3p against MDA-MB-231 and MCF-7. The results highlighted that the conjugated HA-based NPs were able to extend significant targeted cytotoxic effects in the breast cancer cells via activation of the intracellular pro-apoptotic protein p53 and inhibition of surviving. Greater cytotoxicity was observed in case of MDA-MB-231 cells as compared to MCF-7 on account of higher expression levels of CD44 in the former cells than the later [39].

Treatment of colorectal cancer: HA-conjugated or -decorated nanomedicines

Colorectal cancer (CRC), also referred to as bowel cancer, is one of the most commonly occurred cancers with low rate of early diagnosis which ultimately leads to fatal metastatic consequences [1]. Though aside from surgery, chemotherapy with conventional drugs like oxaliplatin and 5-fluorouracil (5-FU) are most commonly employed agents; however, rapidly evolving chemo-resistance has limited their significance. Thus, the quest for development of novel strategies for overcoming drug resistance and improving anticancer efficacy has attracted greater interest of scientists over the past few decades.

Like breast cancer, CD44 receptors are also overexpressed on the surface of bowel tumours. Thus, HA-conjugated NPs have stepped up as a promising solution for the eradication of the barrier associated with chemo-resistance via efficient drug targeting [40]. Nimesulide, an anti-inflammatory cyclooxygenase-2 (COX-2) inhibitor has been reported to extend substantial anti-cancer properties besides having conventional antipyretic and analgesic effects; however, its low hydrophilicity and aqueous solubility undermines its pharmacological significance. To impart hydrophilic biocompatibility as well as targeted delivery, nimesulide was combined with high (360 kDa) and low (43 kDa) molecular weight HA [40]. MRI imaging results demonstrated that both the prepared nanoconjugates were able to extensively penetrate into the CD44-overexpressing HT-29 colorectal cancer cells and lead to effective tumour size reduction within 3 d via apoptosis resulting from RME of HA-conjugated nimesulide, with insignificant morphologic changes observed in kidney and liver [40].

Utilizing specific-targeted ligand ability of HA, HA-tagged/conjugated curcumin-entrapped silica NPs have been engineered for targeted delivery of curcumin and tested against CRCs in vitro and in vivo [41]. The resulting data demonstrated significant reduction in cancer cells proliferation, enhanced cytotoxicity against non-MDR and MDR cancer cells, significantly higher accumulation of curcumin inside colo-320 and HT-29 cells, induction of apoptosis, faster internalization, and reduced tumour volume and metastasis [41].

In an attempt to achieve early cancer diagnosis and efficient targeted delivery of anticancer drugs, an interesting feature was introduced by employing PEG-mediated surface modification of HA-decorated NPs (P-HA-NPs) [42]. As a result of surface modification, P-HA-NPs showed significantly higher selective accumulation in cancerous tissue. In relevance to diagnosis, Cy 5.5 (a near-infrared fluorescence (NIRF) imaging dye was linked with the backbone of HA in P-HA-NPs. Efficient visualization of colon neoplasia was observed upon injection of Cy5.5-P-HA-NPs into bloodstream of tumour-bearing mice, by the use of NIRF imaging (Figure 1). In context to targeted therapeutics, irinotecan (IRT), an anticancer drug, was introduced inside the P-HA-NPs core region. The antitumor efficiency was derived from the fact that IRT-P-HA-NPs showed exceptional suppression of tumour proliferation through tumour-specific cytotoxicity alongside insignificant systemic toxicity, thus exhibiting a promising role in early diagnosis and therapy of colon cancer (Figure 1) [42].

Figure 1. NIRF imaging of liver-implanted CT26 colon tumours. (a) Whole body images of athymic nude mice bearing CT26 colon tumours in their liver tissues after intravenous injection of Cy5.5-P-HA-NPs as a function of time. Arrows indicate the sites of tumours. (b) Ex vivo fluorescence images of tumour-bearing livers and organs excised at 48 h post injection of NPs (5 mg/kg). (c) Representative histological images of normal liver and tumour slices stained with haematoxylin and eosin [42]. Reprinted with permission from American Chemical Society (Copyright © 2011).

For concurrent photodynamic imaging and early diagnosis of cancer, chlorin e6 (Ce6, a photosensitizer) loaded HA-decorated NPs were fabricated via dialysis method [43]. In vitro tests carried out on HT29 cell line revealed that upon administration of Ce6-HANP NPs, followed by Ne–He laser irradiation, significant cytotoxicity was observed with minimal effects on normal cells (NIH313). On the other hand, free laser-treated Ce6 demonstrated higher cytotoxicity in NIH313 cells. Upon intravenous administration inside the HT29 tumour-carrying mice, Ce6-HANPs displayed rapid internalization at the tumour sites and consequently accumulated inside tumour cells via receptor-mediated endocytosis on the basis of interaction in-between HA based NPs and CD44. After NIR laser treatment, Ce6 inside the cells was able to produce fluorescence as well as reactive oxygen species inside cancer cells, leading to significantly suppressed of tumour proliferation [43].

Treatment of pancreatic carcinoma: HA-conjugated nanomedicines

Recent studies have depicted that fatalities associated with the pancreatic cancer are continuously increasing by an annual rate of 0.3% with new cases and associated deaths for 2017 in USA being forecasted to account for more than 53,000 and 43,000, respectively [1]. Pancreatic cancer is also forecasted to move up as the second prime cause of cancer-mediated fatalities in USA and other areas in the world.

For the purpose of achieving optimum cytotoxicity against cancer cells, improved cellular uptake and intracellular penetration of anticancer drugs, enhanced apoptosis of cancerous mass, and targeted delivery of anticancer moieties, several HA-conjugated nanocarrier-mediated systems have been fabricated, characterized and tested against pancreatic carcinoma. These advanced delivery systems include HA-conjugated NLCs [44], NPs [45], graphene quantum dots [46], exosomes [47], dendrimers [48] and liposomes.

Recent report has indicated that NLCs aided co-delivery of baicalein (BCL) and gemcitabine (GEM) possess significant anticancer efficacy against pancreatic cancer [44]. Exploiting the ability of HA to effectively bind with CD44 receptors over-expressed on the cancer cell surface, nano-prodrugs were constructed containing BCL and GEM in the core region and HA bound to the surface [44]. The final HA based BCL-GEM NLCs (HA-BCL-GEM NLCs) were synthesized via nanoprecipitation technique bearing an average diameter of 131 nm and their in vitro cytotoxicity was analysed on pancreatic AsPC1 cancer cells. Results from MTT assay exhibited that the prepared NLCs were effectively accumulated into the cancer cells where upon they demonstrated significantly higher cytotoxicity compared to the control groups on account of HA-CD44 based interaction. Furthermore, in vivo results showed effective tumour proliferation inhibition upon treatment with HA-BCL-GEM NLCs against murine C65BL/6 pancreatic cancer model [44].

CD44 over-expression on the surface of cancer cells causes stimulation of several oncogenic cell pathways such asAkt-Pi3K-NF-kB pathway responsible for cancer malignancy and progression. Thus, HA-conjugated PLGA-PEG NPs were developed for TTQ (thio-tetrazolyl derivative of a candidate molecule, IC87114) targeted delivery to CD44 positive pancreatic cancer cells [45]. These NPs were synthesized via solvent evaporation method and the size of the final HA-modified construct was calculated to be <200 nm, exhibiting a spherical morphology. In vitro cellular uptake and cytotoxicity studies revealed that these HA-conjugated NPs demonstrated greater intracellular penetration and specific cytotoxicity in comparison to PEG-PLGA NPs in CD44 positive MiaPaca-2 cells via modulation of Akt-Pi3K-NF-kB pathway [45].

Recently, a novel nanoformulation was explored comprising of graphene quantum dots (GQDs) nanoconjugates for pancreatic bio-imaging and targeting of anti-neoplastic drugs [46]. The success of this new nanoformulation was established by showing pronounced cytotoxicity against pancreatic cancer cells in vitro (Panc-1), enhanced internalization of anti-neoplastic agents, significantly higher bioavailability, marked penetration and uptake, reduced cancer tissue mass and anticancer efficacy [46].

Endosome-derived small intraluminal vesicles called “Exosomes” are important molecules that help in promoting intercellular communication via delivering contents, for example nucleic acids and proteins amongst cells that are adjacent in microenvironment of tumour. The study aimed at deciphering the mechanisms by which cellular communication between macrophages and human pancreatic cancer cells occur through exosomes-mediation [47]. Upon exosome-based cell communication characterization, transfection of pancreatic cancer cells Panc-1 was carried out via plasmid expressing miRNA-155 and miRNA-125b2 employing self-assembling HA-polyethylene imine/HA-PEG NP-derived non-viral vectors. Results exhibited that following Panc-1 cells transfection, an alteration in the exosomal content was observed, causing reprogramming and differential communication of cells of J774-A1 to M-1 phenotype. These results established that gene-mediated targeted therapeutics against specific manipulation of cancer cell-based exosomal content can play a promising role in pancreatic cancer therapy [47].

Utilizing the selectively binding properties of HA to CD44 receptors overexpressed on the surfaces of pancreatic cells, Kesharwani et al. [48] fabricated micelles and poly-amidoamine-dendrimer carrier (PAMAM) for the targeted delivery of anti-neoplastic agents. Results depicted that conjugation of HA as molecular ligand on the surface of these delivery systems significantly improved cytotoxicity against CD44-overexpressing pancreatic cell lines (AsPC-1 and MiaPaCa-2). They have also noticed significantly higher uptake and cellular internalization, increased apoptosis of anticancer cells and enhanced anticancer efficacy [48].

Treatment of lung cancer: HA-conjugated nanomedicines

Lung cancer is another endemic cancer sub-type with 22,500 new cases and associated 155,870 fatalities for the year 2017 in USA alone. Studies have shown that lung cancer accounts for more than 26% of all deaths affiliated with cancer [1]. In order to reduced fatalities and death rate associated with lung cancer; many researchers attempted the delivery of anticancer moieties using nanodelivery systems such as HA-conjugated mixed micelles [49], NPs [50], NLCs [51], nanocages [52], PLGA NPs [53], GO-NPs [54] and nanocomposites [55].

Baohuoside I, a flavonoid derived from Herba epimedii, has been reported to exhibit excellent chemotherapeutic properties but its hydrophobic nature undermines its pharmacological uses. Recently, mixed micelles derived from baohuoside I paired-D-a-tocopheryl polyethylene glycol succinate (TPGS) and dodecyl-dimethyl-ammonium bromide (DDAB), conjugated with the targeting moiety HA were engineered for imparting hydrophilicity, stability and limited efflux of the drug from lung cancer cells [49]. This study was carried out for determining the in vitro cellular accumulation and affiliated anti-proliferative and anticancer effects, in addition to cancer targeted delivery evaluation employing A549 cells in tumour xenograft mice models. Enhanced level cellular uptake alongside significantly lowered IC₅₀ concentration was displayed by the prepared micelles in contrast to free drug. Moreover, increased antitumor activity was also exhibited by the prepared micelles in vivo, demonstrating the potential of the micelles for use as effective anticancer agents in lung cancer [49].

Interestingly, HA has also been successfully utilized as targeting ligand in photodynamic therapy (PDT) [50]. In this study, chlorin e6 (Ce6), a NIR photosensitizer was loaded into the PLGA NPs conjugated with HA for targeting CD44 receptors on surface of lung cancer cells (A549 cells). HA carboxylate groups facilitated the binding of MRI contrast agent gadolinium ions (Gd3+) onto the outer layer of PLGA NPs. The prepared formulation was shown to effectively target CD44-positive A549 cells, as confirmed via MRI and fluorescene results in vitro and in vivo. Upon irradiation of the tumour xenograft A549 previously treated with the NP constructs, a significant reduction of tumour proliferation and consequent regression was observed, suggesting the promising potential of the HA conjugated multifaceted NPs in MIR imaging and PDT against lung cancer [50].

Besides PTD, HA has also been used as targeting ligand for the tumour-specific delivery of anti-neoplastic drugs for treatment of lung cancer in the form of NLCs [51] and nanocages [52]. Results have shown significantly higher internalization by CD44 over-expressing subcutaneous A549 carcinoma cells, greater haemocompatibility and biocompatibility, reduced IC_50, dose-dependent cytotoxicity and apoptosis in cancer cells (A549 cells), reduced tumour volume, significant reduction in the expression of luciferase and superior antitumor efficacy [51,52].

The success of HA-conjugated nanodelivery system against orthotopic human lung cancer has been demonstrated by Wu et al. [53]. In this study, lung tumour-specific targeting of docetaxel (DTX) was attempted using HA-conjugated PLGA NPs. Physicochemical analysis revealed that these NPs (DTX-HPLGA) were bearing a diameter of 154 nm. A significantly lower IC₅₀ values was observed in contrast to the free DTX and a significantly higher (approx. 4 folds) uptake into the A549 lung cancer-bearing mice. Furthermore, in this study, tumour treating ability of DTX-HPLGA was evaluated in orthotopic human A549-Luc lung tumour xenografts [53]. Resulting evidence showed a weaker tumour bioluminescence in mice group treated with DTX-HPLGA compared to free DTX throughout the treatment period of 16 d. These results evidenced that DTX-HPLGA can suppress tumour growth effectively. The semi-quantitative analysis of radiance further corroborated that DTX-HPLGA caused significantly more effective tumour inhibition than free DTX. The bioluminescence imaging of lung tissues isolated on day 16 confirmed that mice treated with DTX-HPLGA had the weakest tumour luminescence and invasion among all the treatment groups [53].

For overcoming MDR development, a novel carbon nanomaterial Q-Graphene was prepared as an effective therapy against lung cancer cells [54]. Both the attributes of fluorescence imaging and targeted delivery were installed in the prepared NPs by conjugation with rhodamine B isothiocyanate (RBITC) and HA. DOX was used as the preferred anticancer drug in the said model and exhibited 86% drug entrapment efficiency (DEE). On account of the RBITC probing, the accumulation of NPs and release of intracellular DOX could be visualized within MRC-5 and A549 lung cancer cells. Thus, effective monitoring of targeted delivery of drugs could be ensured for eradication of MDR lung cancer cells [54].

Another attempt to prevent occurrence of MDR was made by Ganesh et al. [55]. They argued that the targeted delivery of siRNAs which cause down-regulation of over-expressed anti-apoptotic genetic molecules in drug resistant tumour tend to exhibit synergistic anticancer effect upon co-administration of cisplatin (CDDP)-loaded HA based NPs against CD44 positive lung cancer cells. Various results were obtained from confocal microscopy, PCR and mass spectrometry (ICP-MS), following IV administration of HA-based drug loaded NPs using in vivo A549 tumour xenograft bearing mice models. They demonstrated that CDDP and siRNA distribution pattern employing CD44 specific HA NPs correlated impressively with the cancer-targeting ability and efficacy demonstrated with combinational treatments [55].

Treatment of skin cancer: HA-conjugated nanotherapeutics

The estimated occurrence of skin cancer and associated deaths has been forecasted to rise to more than 95,000 and 13,590 for the year 2017 in the US [1]. Amongst its subtypes, malignant melanoma is the most aggressive type. Unfortunately, drug resistance associated with the conventional chemotherapies has posed as a major challenge, making the research for novel therapeutic drugs against skin cancer a prime necessity.

The quest for development of novel therapeutic strategies for treatment of melanoma led to preparation of polymer-based photo-thermal agents derived from poly-N-phenylglycine (PNPG), (prepared though N-phenylglycine (NPG) polymerization) for employment in NIR-mediated-phototherapy and generating ROS upon irradiation [56]. PNPG was further conjugated with HA as a targeting ligand and PEG-diamine as a potential coupling agent. HA-conjugated PNPG (HA-PEG-PNPG) demonstrated an effective targeting to CD44 over-expressing B16 melanoma cells. In vitro and in vivo results demonstrated that HA-PEG-PNPG selectively induces apoptosis in B16 cells whilst suppressing tumour proliferation in malignant melanoma upon irradiation with NIR at a wavelength of 808 nm, exhibiting the promising potential of the HA-based nanoformulation for targeting PDT of melanoma [56].

For the purpose of enhancing uptake of anticancer drugs within CD44 positive B16F10 skin cancer cells, oligomeric HA conjugated tumour-penetrating peptide iRGD-based liposomes have been engineered [57]. The prepared conjugates were loaded with DOX for the reduction of tumour growth in mice and enhancing tumour apoptosis. Results highlighted that DOX-loaded HA-based liposomes were readily up-taken by the skin cancer cells, and this penetrating ability was further synergized by loading the liposomes with iRGD. In contrast to the control group in which HA was not conjugated onto the drug loaded liposomes, HA-linked liposomes were able to substantially internalize within the cells on account of CD44-HA interaction and demonstrate profound anticancer activity, thus exhibiting a promising role for the treatment of melanoma [57].

Another nanodelivery system comprises of calcium phosphate hybrid NPs bearing siRNA was prepared [58]. These hybrid NPs were further thiolated with HA as a targeting ligand and smart redox-simulative delivery vehicle. These smart hybrid NPs were effectively able to deliver siRNA into the cancer cells via CD44-HA based endocytosis leading to approximately 80% silencing of Bcl-2 and luciferase genes. The gene silencing led to induction of apoptosis in B16F10 skin cancer cells. Together with their ability to achieve efficient drug targeting to cancer cells, these NPs also reduced the proliferation of tumour xenograft B16F10 mice with minimal non-specific cytotoxicity, thereby showing potential to be used as novel anti-cancer delivery system [58].

Adopting the CD44 targeting ability of HA, Shen et al. [59] developed HA-conjugated SLNs encapsulated PTX, directed against B16F10 melanoma cancer cells. The resulting SLNs, bearing an average size of 190 nm, exhibited a dose-dependent cytotoxicity in CD44 + B16F10 cells in vitro as revealed via MTT assay. Using annexin V stain, substantial level of apoptosis was also observed in the NPs-treated cells. Results from invasion assay and adhesion assay using Boyden chamber and Matrigel revealed that these SLNs were able to cause significant inhibition in both cases in a dose-dependent fashion. Significant anti-tumour activity was also observed in the B16F10 xenograft C57BL/6 mice with minimal histopathological toxicity in comparison with free drug [59].

For the treatment of melanoma, a novel HA-nano-GO conjugates (HA-NGO) were fabricated for NIR laser mediated photo-thermal ablation therapy [60]. Confocal microscope aided approach revealed that the prepared nanoconjugates were efficiently delivery to the tumour site in mice via transdermal delivery, believed to have been caused via interaction with the HA receptors on the surface of tumour cells. Irradiation with NIR not only caused eradication of tumour tissues but also prevented future recurrence. The antitumor activity was evaluated via reduced caspase-3 activity obtained by ELISA assay and immune-histochemical and histological findings using TUNEL assay for cancer cell apoptosis. The results confirmed the appropriateness of HA-NGO transdermal delivery for photo-thermal-ablation therapy in melanoma [60].

To induce higher potential of cytotoxicity and apoptosis in cancer cells, a unique anticancer approach was adopted by Montanari et al. [61] using targeted delivery of polyamine degrading enzyme, bovine serum amine oxidase (BSAO), an enzyme that converts polyamines over-expressed in malignant cells into hydrogen peroxide and aldehyde(s), thus inducing high cytotoxicity in cancer cells. The biosynthesis of polyamines (such as spermine) tends to cause proliferation of cancer cells whereas its degradation causes inhibition. BSAO tends to degrade polyamine followed by release of cytotoxic ROS bi-products such as hydrogen peroxide. Conjugating of BSAO with self-assembling HA-cholesterol nanohydrogels (HA-CH-NH) not only imparts stability but also targeted delivery towards M14 melanoma cells. The results exhibited that the prepared HA-conjugated nanohydrogels exhibited targeted delivery of BSAO via HA-CD44 mediated endocytosis lead to significant cytotoxicity in cancer cells via enhanced generation of ROS through polyamine degradation [61].

Owing to the promising potential of carbon nanotubes (CNTs) in drug delivery and photo-thermal therapeutics, HA-conjugated-CNTs were designed for effective tumour targeting against B16F10 cells and a novel photodynamic therapy (PDT) agent, haematoporphyrin monomethyl ether (HMME), was loaded onto the surface of HA-based CNTs to fabricate HA-HMME-CNTs [62]. The synergistic activity of PDT and photo-thermal therapy (PTT) against tumour growth was analysed both in vitro and in vivo after administration of HA-HMME-CNTs. Significant cancer therapeutic activity, as revealed via enhanced apoptosis and tumour growth inhibition, was observed after dual treatment of PDT and PTT, in contrast to individualized treatment, with insignificant toxicity towards normal organs [62].

Retinoic acid has been reported to prevent cellular proliferation and has the ability to be taken up by the nucleus inside the cells. Thus, all trans-retinoic acid (ATRA)-HA conjugates were designed for targeted delivery of the anticancer drug PTX, inside the B16F10 melanoma cells [63]. Results revealed that the prepared nanoconjugates were haemocompatible, extensively taken up by the CD44 + cancer cells and were translocated in to the nucleus leading to enhanced apoptosis as revealed by annexin V staining. In vivo results from B16F10 tumour bearing C57BL/6 mice revealed that these nanoconjugates were extensively accumulated inside the tumour and displayed a significant reduction in the tumour size in contrast to free PTX [63].

Treatment of ovarian carcinoma: HA-based nanomedicines

Ovarian cancer is an evasive and fatal syndrome, bearing the greatest mortality percentage amongst all types of female reproductive cancers [1]. On account of minor clinical symptoms at the inception, majority of the patients are diagnosed with metastasized disease during diagnosis. Despite chemotherapeutic and surgical strategies, a very low rate of survival is associated with these patients. Hence, novel therapeutics designs are desired for effective eradication of the said disease.

Prevalence of MDR associated with ovarian cancer is rapidly increasing and has limited therapeutic efficacy of anticancer drugs. Over-expression of genes like MDR1 and P-gp is deemed as major hallmarks associated with the problem. To overcome the said problem, MDR1 targeted siRNAs have stepped up as a promising solution however, systemic stability and targeted delivery pose up as a major hurdle in its prolific application. Therefore, HA-PEG/HA-PEI was fabricated for delivering MDR1 siRNA, followed by administration of PTX for suppression of ovarian cancer growth [64]. HA-PEG/HA-PEI effectively delivered MDR1 siRNA to the cancer site on account of HA-CD44 interaction, and caused substantial down-regulation of P-gp and MDR1. This was followed by administration of PTX which caused substantial inhibition of tumour growth and induction of apoptosis as the MDR driving mechanisms were down-regulated by the previous application of HA-PEI/HA-PEG MDR1 siRNA. Thus, this strategy can be employed as a prolific tool for treatment of ovarian cancer [64].

Moreover, studies reporting the acidic microenvironment inside the tumour mass have led to development of pH-sensitive drug delivery vehicles. Liu et al. [65] thus fabricated pH-responsive HA-modified TiO2 NPs for targeted delivery of CDDP against A2780 ovarian cancer. Difference in pH responsive drug release rate was observed by finding merely 22% drug release at pH of 7.4 which enhanced to 90% at acidic pH (pH 5.0). Results from florescence microscopy revealed that the CDDP-loaded HA-modified NPs were readily up-taken by the cancer cells on account of CD44-HA interaction and caused significantly enhanced cytotoxicity compared to control group. In vivo results from A2780 tumour xenograft mice also showed that HA-conjugated NPs were readily up-taken by the tumour cells, causing minimal toxicity in the surrounding tissues [65].

To optimize the bioavailability and uptake into tumour cells, inducing apoptosis, minimizing generalized toxicity and drug resistance, and reducing tumour volume and tumour growth, PTX-loaded NLCs have also been engineered [66].

Treatment of cervical cancer: HA-conjugated nanomedicines

Cervical cancer is the second most common type of cancer amongst middle-age women in the US with approximately 13,000 new cases forecasted in the year 2017 [1]. As one of the oldest recognized cancers, an optimal therapeutic regimen is yet to be designed for the eradication of the said menace. HA-conjugated nanomedicines have stepped up as a promising solution for treatment of cervical cancers.

In an attempt to design multipurpose nanoconjugates, Prussian blue (PB) NPs were designed as carrier and photo-thermal agent, HA grafting PEG (HA-g-PEG) as capping and CD44 receptor targeting agent, and 10-hydroxycamptothecin (HCPT) as oncotherapeutic [67]. HCPT is an alkaloid derived from Camptothecaacuminata Decne and has been reported to demonstrate significant activity against cervical carcinoma. The final HCPT-loaded NPs were highly stable, biocompatible and histologically non-toxic and effectively targeted CD44 over-expressing HeLa cells [67]. These nanocarriers further showed substantial photo-thermal activity and a light-stimulated release of HCPT. Results from in vivo studies carried out on HeLa xerograft bearing nude mice showed that the prepared NPs were able to significantly reduce the size of tumours via synergistic effect of chemo and photo-thermal therapy respectively, with minimal general toxicity [67].

For overcoming limitations associated with conventional PTT, such as non-specific activity and hyperthermia of normal tissues, Jiang et al. [68] have prepared HA-conjugated polyaniline NPs (PANI-HA NPs) by one-step oxidation polymerization mechanism and directed against CD44 + HeLa cancer cells. Result from confocal microscopy and MTT assay revealed that PANI-HA NPs were readily up-taken by the cancer cells but administered ROS-mediated cell death upon exposure to 808 nm NIR radiation. Moreover, a substantial reduction in the size of tumour size was observed in mice treated with HA-conjugated PANI-HA NPs [68]. For optimum tumour-specific delivery, minimized generalized toxicity, abolishing proliferation of tumour cells and growth, and improved anticancer efficacy, HA-conjugated GO-based DOX-encapsulating NPs, hybrid hydrogels and Ce6 photosensitizer tagged NPs have also been engineered for treatment of cervical carcinoma.

Treatment of head and neck cancers: HA-based nanoconjugates

Head and neck cancer tends to be the sixth most prevalent cancer type worldwide, with squamous cell carcinoma (SCC7) accounting for nearly 90% of the associated cancers and recognized as a recurring aggressive malignancy [1]. As CD44 receptor is over-expressed on SCC7 surface, novel HA-coupled/linked nanomedicines have been designed for treatment of these types of cancers.

Though exhibiting promising anticancer efficacy, optimal use of PTT is hampered due to lack of targetability. For effective transport of PTT agents to the tumour site, a multifunctional nanoformulation composed of the photoacoustic imaging (PA) and PTT agent copper sulphide (CuS) loaded onto Cy5.5-based HA NPs was prepared [69]. The CuS tends to quench the fluorescent signal of Cy5.5 in nanoformulation, but upon HAase-mediated degradation inside tumour environment, a strong fluorescent signal is produced. Upon IV administration of CuS-HANPs in SCC7 xenograft tumour-bearing mice, high PA signals and fluorescence were obtained in the tumour environment, which reached its peak after 6 h. Afterwards, the tumours were subjected to laser-irradiation, which lead to significant inhibition rate of tumour (89.74%). The results encouraged the employment of HA-conjugated multifunctional nanoformulation for image-based PTT in chemotherapeutic applications [69].

For tumour-specific delivery of DOX and PTX against squamous cell carcinoma (SCC-7), HA-conjugated poly-caprolactone copolymers (HA-PCL) and cholanic acid micelles (HA-CA) have also been engineered [70]. Results clearly evidenced superior targetability to SCC7 cells and substantial reduction in tumour volume. Also the enhanced bio-distribution levels of PTX-HA-CA conjugates were observed into the cancer cells of SCC7 xenograft mice. This targeting potential was attributed to HA-CD44 interaction resulting in RME. The size of the tumour mass was significantly decreased (540 mm³) in a dose-dependent fashion with no apparent side effects in contrast to free PTX (1071 mm³) and control group (883 mm³) at day 8 (Figure 2). Interestingly, a follow-up analysis revealed that the size of the tumour was increased from day-8 to onward after the treatment with PTX-loaded micelle was stopped, indicating a remarkable anticancer efficacy of PTX-encapsulated micelles in alleviating tumour progression [70].

Figure 2. In vivo tumour inhibition study. Balb/c nude mice were injected with PBS, free paclitaxel and HA-CA-Paclitaxel at Day 0 (n = 3). Comparison of tumour size on Day 8 of (A) Control (B) Paclitaxel (C) HA-CA-Paclitaxel (2 mg/kg) (D) HA-CA-Paclitaxel (5 mg/kg). Tumour inhibition data show mean tumour volume, of triplicate samples ± SD. *p < .05 relative to HA-CA-paclitaxel (2 mg/kg) and **p < .05 relative to control group [70]. Reprinted with permission from Elsevier B.V. (Copyright © 2014) through Copyright Clearance Centre.

For the purpose of diagnosis and monitoring of cancer, HACE-inspired nanoprobes have been engineered for optical and MR imaging [71]. The HACE was chelated with gadolinium (Gd), used as MR contrast agent via diethylene-triamine-pentaacetic dianhydride (DTPA). Cy5.5 was additionally bonded onto the backbone of HACE, acting as a NIR fluorescene dye. The final nanoconjugates (Cy5.5-DTPA-Gd-HACE) demonstrated higher biocompatible and were extensively up-taken by CD44 + SCC7 cells in contrast to U87-MG cells with low receptor expression, highlighting CD44-HA based interaction governing the cellular accumulation. The increase in MR contrast of HACE-based probe was also confirmed in SCC7 xenograft tumour mice model. Targetability of the nanoprobe towards the tumour was evaluated via NIRF imaging, and enhanced accumulation of the HACE-based nanoprobe was observed in the tumour region. The studies reveal that dual-imaging mediated by HACE-based nanoformulation may be employed for accurate monitoring and diagnosis of head and neck cancers [71].

Treatment of gastric cancer: HA-based nanomedicines

Gastric cancers (GCs), specifically the subtype with enhanced CD44 expression, is notoriously known as the fourth most prevalent cancer type and the second-major cause of cancer associated fatalities globally. The five year survival rate is reported to be nearly 15% on account of metastasis, rapid proliferation and recurrence [1]. To optimize target-specific delivery of anticancer drugs and to improve their therapeutic efficacy, various nanomedicines including NLCs [72], nanotubes [73] and nanoplexes have been employed.

The NCCN (National Comprehensive Cancer Network Purpose) recommended the adoption of combined chemotherapy for the treatment of GCs. However, generalized non-specific toxicity poses major concern and undermines its optimal usage. For overcoming the non-target, generalized toxicity, Qu et al. [72] designed NLCs for the combined delivery of two chemotherapeutic drugs, CDDP and 5-FU. Anti-cancer cytotoxicity studies carried out on BGC823 human GC cells demonstrated a synergistic effect with the dual-drug therapy and displayed a significantly enhanced antitumor effect in contrast to the free form of drugs [72].

Another novel nanodelivery system, single wall carbon nanotubes (SWNTs), was designed by Yao and coworkers [73] against CD44 + gastric CSCs. For targeted delivery, the nanotubes were further conjugated with HA and chitosan (CHI) and encapsulated with salinomycin (SAL). These nanotubes (HA-SAL-CHI-SWNTs) effectively inhibited the self-renewal ability of the CD44 + cells and reduced mammosphere and colon development of CSCs. Furthermore, the invasive and migratory capacity of GC CSCs underwent significant blockage after treatment with HA-SAL-CHI-SWNT. Cellular uptake studies revealed that HA-conjugated facilitated rapid accumulation of the HA-SAL-CHI-SWNT into the cancerous cells, whereas pre-application of free HA resulted in inhibition of uptake, highlighting the involvement of CD44-HA mediated endocytosis mechanism. These nanotubes hence exhibited profound antitumor activity against GC CSCs and mammospheres via induction of apoptosis and deep penetration inside the core region, respectively. Hence, the use of HA-SAL-CHI-SWNT nanotubes holds promise in the eradication of GC CSCs [73].

Anticancer efficacy of HA-conjugated nanomedicines: Clinical studies

A multitude of HA-based nanomedicines have acquired the status of Phase 2 and Phase 3 clinical trials on account of their promising characteristic attributes in cancer treatment. These novel formulations are generally being used in injectable form and as polymer-based drug conjugates. An obstacle in the employment of HA-based nanoconjugates in the area of oncotherapeutics is their grouping under new chemical entities (NCE); hence the physiochemical characteristics, such as particle size, morphology, drug loading capacity and release profile need to be subjected to extensive optimization.

Though the recent clinical profiles have revealed a marked reduction in the anticancer regimen associated side effects; however, their tumour-specific targeting and anticancer efficacy require substantial improvement. To this end, suitable linkers to fine tune drug release will be mandatory to enhance the therapeutic outcomes further. A series of chemotherapeutic drugs, such as methotrexate (MTX), DOX, IRT, CPT, CDDP, DTX, etc., have undergone extensive investigation in clinical and preclinical trials via utilization of HA as a targeting ligand. These preclinical findings highlighted the ability of HA-based nanoconjugates to exhibit modest anticancer activity and superior safety profiles in contrast to conventional anticancer regimen. Results from Phase-1 clinical trials, carried out on 12 patients, revealed the preparation of IRT-HA to be substantially safe and easily tolerated with no compromise on IRT anti-cancer activity [74].

Furthermore, another phase 2 clinical trial was carried out on 41 patients, highlighted the benefits of HA-based nanoconstructs with regards to the progression-free safety and survival [75].

In 2014, Alchemia Oncology conducted a phase 2 clinical trial (NCT02216487) involving 5-FU and IRT-HA plus cetuximab. The objective of the trial was to understand the use of IRT-HA based on a single-arm experiment covering (HA)-FOLF and cetuximab amongst second-line IRT-naïve patients bearing KRAS wild-type CRC. Additionally, the study aspires to confirm efficacy and safety profile of (HA)-FOLF and cetuximab for use in second-line treatment amongst IRT-naïve patients suffering from metastatic CRC. Later in 2015, a phase 1 clinical trial was carried out for elucidating the antitumor effect of PEGylated recombinant HAase in conjugation with DOC targeted against previously-treated recurring metastatic lung cancer.

Furthermore in 2016, other randomized phase 1 and 2 clinical trials (NCT02753595) were conducted for studying the comparative anticancer effect of eribulin mesylate encapsulated within PEGylated recombinant HAase against free eribulin mesylate in patients suffering from hyaluronan over-expressing metastasized breast cancer. Two other studies also commenced in the same year. One (NCT02715804) aimed at the determination of anticancer effects of PEGylated recombinant HAase combined with Nab-PTX and GEM compared to placebo and Nab-PTX plus GEM in patients with untreated HA-over-expressing stage 4 pancreatic adenocarcinoma. The other (NCT02910882) involving the study of combined PEGylated recombinant HAase and GEM along with radiotherapy in participants suffering from pancreatic cancer.

Conclusions

Hyaluronic acid-based conjugation or modulation of various anticancer drug-loaded nanocarriers including NPs (polymeric and metal), liposomes, micelles, SLNs, NLCs, nanoplexes, nanohydrogels, nanogels, nanomaterials, nanocomposites, nanoconjugates and carbon nanotubes have shown modest improvement in anticancer efficacy. Utilizing the tumour-specific targetability of HA molecule, the generalized toxicity and off-target effects related to the chemotherapy can be minimized; thereby, maximizing therapeutic outcomes. The critical analysis of the literature revealed that hyaluronic acid-modified nanoconjugation has been emerged as an efficient approach for cancer imaging and anticancer regimen against CD44 over-expressing tumours. Findings demonstrated their ability to rapidly accumulate inside the tumour cells via CD44–HA endocytosis and RME mechanisms, inhibition of proliferation and growth of tumour cells, induction of cancer-specific apoptosis and prevention of metastasis, especially in case of MDR tumours as well as improved bioavailability and anticancer efficacy. Furthermore, hyaluronic acid based nanoconjugates have been proven stable, biocompatible, and haemocompatible and thus appropriate for further use in clinical trials.

Future prospects

Nevertheless, plethora of in vitro, in vivo and clinical studies have explored the pharmaceutical implications and therapeutic applicability of HA-conjugated/modified nanotherapeutics to achieve rapid internalization of anticancer moieties into the tumour cells, to induce cancer-specific apoptosis, to down-regulate proliferation and differentiation of cancerous cells and metastasis; however, much has yet to be explored. Substantial gaps in the research have been identified which include but not limited to; (1) lacking of comparative analysis of various nanomedicines for anticancer efficacy and reduced MDR resistance, (2) lacking of ample safety data for the nanomedicines carrying anticancer payloads, (3) lacking of adequate evidence-based randomized research specifically exploring the therapeutic roles of HA-conjugated nanotherapeutics in reducing the non-target adverse events and (4) lack of insight into the large-scale (industrial) manufacturing of nanoencapsulated HA-based therapeutics. These future prospects will provide deep insights to the scientists and researchers working in the field of cancer therapies to further elucidate and comprehend the pharmaceutical significance of nanomedicines in achieving target-specific delivery of cancer therapeutics and preventing the development of MDR, chemo-resistance, cancer metastasis and improving rationalization in cancer therapy.

Disclosure statement

The authors report no conflict of interest in this work.

Funding

The authors would like to acknowledge Universiti Teknologi MARA for providing LESTARI grant (600-IRMI/DANA 5/3/LESTARI (0007/2016)) and support in writing this review article.