Hyaluronic Acid Based Drug Delivery Systems for Cancer Therapy

1. Introduction

One of the most pressing problems currently facing pharmacology is the development of new chemotherapeutic drugs. The proportion of malignant tumors sensitive to chemotherapy is quite small. Alternative treatments, such as surgery and radiation therapy, are not always effective, especially at the metastasis stage. Cytostatic drugs often have many side effects, including nephrotoxicity, polyneuropathy, myelosuppression, ototoxicity, allergic reactions, dyspepsia and cardiovascular disorders. The development of drug delivery systems is aimed at improving the effectiveness of the drug without increasing side effects [1,2].

One of the modern and actively developing areas at the intersection of chemistry, pharmacology and oncology is supramolecular chemotherapy, based on the possibility of forming complexes of the “host-guest” type [1,2,3,4]. Macrocyclic compounds such as cucurbiturils, cyclodextrins, calixarenes, etc., are ideal platforms for constructing chemotherapeutic platforms through supramolecular interactions, since they contain cavities of controlled size into which “guest” molecules can penetrate [5,6]. Supramolecular chemotherapy, which combines non-covalent interactions and traditional chemotherapy, has demonstrated a number of advantages, for example, the ability to increase the solubility and stability of drugs, reduce side effects, and can also be used for targeted drug delivery. As is known, the essence of targeted delivery is that the drug itself, and more often the means of its delivery (vector, container) are modified by molecules that recognize structures on target cells. In the case of supramolecular delivery systems, hyaluronic acid can both serve as a ligand and act as a molecular framework [7,8]. Since hyaluronic acid is not a bioinert molecule, when creating drug delivery systems based on hyaluronic acid, it is necessary to take into account its role under normal conditions and in oncology.

2. Hyaluronic Acid

Hyaluronic acid (HA) is a natural polysaccharide that is widespread in the human body and is of great importance for its physiological processes [9]. HA is one of the key components of the extracellular matrix and is found in various tissues and structures, mainly in connective tissue, joints, epidermis and the eyeball. Due to its high hydrophilicity, HA has unique properties, such as the ability to form viscous gel-like structures, retain moisture, provide lubrication of joints, improve elasticity and firmness of the skin and tissue hydration, and actively participate in the regeneration of damaged tissues [10,11,12].

Hyaluronic acid is a glycosaminoglycan formed by repeating dimers of the disaccharide N-acetylglucosamine and glucuronic acid (Figure 1). The unique characteristic of this polysaccharide is its lack of association with protein structures and the absence of sulfated groups, which distinguishes it from other glycosaminoglycans.

Hyaluronic acid (HA) is an important component of the intercellular space of many tissues in the body. In addition to its reinforcing and buffering functions, HA is an important source of information about the environment for cells. The key factors in this information are the number of cellular contacts with HA via HA-binding receptors, as well as the length of the bound hyaluronic acid fragment. [13,14]

The most important information from the environment for a cell is information about the integrity of the tissue of which it is a part. Thus, the integrity of the tissue, and therefore the components of the extracellular matrix (ECM), which include HA, can be disrupted during wound injury, inflammation, and tissue remodeling. During these effects, the number of cell contacts with the ECM decreases, and the ECM themselves can be destroyed with the formation of oligomers. [15,16,17] Both of these factors inform the cell of the need for an adaptive response to the current conditions, which on a tissue scale is expressed in its remodeling. Namely, in the proliferation of cellular components, increased production of ECM, testing of tissue cells for “professional suitability” (suitability for proliferation), migration of specialized cells to control and restore tissue function, as well as renewal of vascularization of new tissue. [13,18]

HA is a ligand for such receptors as CD44, RHAMM (Receptor for Hyaluronic Acid Mediated Motility), lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), hyaluronan receptor for endocytosis (HARE/stabilin-2), LAYN and Toll-like receptors (TLR) [19,20].The interaction of HA with the CD44 receptor activates key intracellular signaling pathways, which increases the expression of several genes responsible for proliferation and cell survival. This interaction also induces the reorganization of the actin cytoskeleton, which leads to active cell migration.

Tumors, as a pathological type of tissue remodeling process, use the same logic and signaling pathways for tissue degeneration. The role of HA in oncology is based mainly on data from experiments on the CD44 receptor regulation system [13,21,22] It is expressed on many cells in the body and its regulation is often of decisive importance in in vivo experiments in animal models. Nevertheless, there are tumors that are CD44-dependent, CD44-weakly dependent or CD44-independent, such as Ewing’s sarcoma. Other HA-binding receptors may also come to the fore in the regulation of such tumors. Data on their isolated and synergistic effect with CD44 were obtained on cell cultures.

In the literature, it is common to divide HA chains into functional groups depending on the length of the polysaccharide: oligo-HA (< 10 kDa), low molecular weight 10-200 kDa (L-HA), medium molecular weight 0.25-1 MDa (M-HA) and high molecular weight more than 1 MDa (H-HA) and HA with very high molecular weight (VH-HA, > 6000 kDa) [18].

The interaction of receptors with HA depends on the molecular weight of HA. HA with a higher molecular weight has high avidity for CD44 and can lead to their clustering on the membrane surface, thereby modulating the cellular response. H-HA can form steric protection on the membrane surface, limiting access to cell death receptors, protecting cells from apoptosis [14,18].

Low and medium molecular weight HA stimulates the synthesis of heat shock proteins, demonstrating antiapoptotic, proangiogenic and immunostimulating properties [13,14,18,21]. Experiments demonstrate that L-HA has a proinflammatory effect and can activate mouse alveolar macrophages or cause phenotypic maturation of human dendritic cells. It was found that L-HA increases the expression of cytokines such as MMP-12, plasminogen activator inhibitor-1, macrophage inflammatory protein (MIP)-1α, MIP-1β, monocyte chemoattractant-1, keratinocyte chemoattractant, interleukin (IL)-8 and IL-12 in macrophages. Stimulation of CD44 by L-HA leads to the activation of 2 tyrosine kinases. Activation of p185HER2 promotes cell growth, while c-Src kinase activity results in phosphorylation of cytoskeletal proteins, thereby inducing cell migration.

3. Hyaluronic Acid and Oncology

Such optimal properties as low toxicity, high biocompatibility, biodegradation, stability, hydrophilicity and bioadhesion have allowed the wide use of hyaluronic acid to create delivery systems for antitumor drugs. However, the properties of HA itself in relation to various types of cancer are often not taken into account when using targeted delivery systems. Numerous recent studies have proven the role of hyaluronic acid in the proliferation, invasion, apoptosis and dormancy of tumor cells [19]. L-HA exhibits pro-inflammatory, pro-angiogenic properties, stimulates tissue damage and cancer metastasis. H-HA is characterized by anti-inflammatory, anti-angiogenic, antioxidant properties [23,24,25].

Hyaluronic acid is an important component of the extracellular matrix in both normal tissues and tumors. Being the basis of the extracellular matrix, HA can affect various cells of the tumor microenvironment. The role of the tumor microenvironment in carcinogenesis is currently assessed on a par with oncogenes, antioncogenes and the accumulation of mutations in DNA. There are studies in which a high frequency of driver mutations was detected, but at the same time the normal structure and functioning of more than a quarter of the cells was preserved [26]. Such studies only confirm the full value of the tumor microenvironment in the origin and development of a tumor. The composition of the tumor microenvironment is represented by fibroblasts, tumor-associated fibroblasts, myofibroblasts, endotheliocytes, pericytes, neutrophilic and eosinophilic leukocytes, etc. [27]. HA fragments obtained by cleavage with hyaluronidase diffuse through tissues and bind to HA receptors on peripheral cells, acting as intercellular signaling molecules. During the progression of malignant neoplasms, the rates of HA biosynthesis and degradation are significantly increased relative to normal tissues. This leads, on the one hand, to increased formation of the extracellular matrix and high-molecular HA, and on the other hand, to HA fragments. The extracellular matrix, with its high ability to retain moisture, creates optimal conditions for the proliferation and migration of tumor cells [28], and HA fragments stimulate the growth of endothelial cells, angiogenesis [29]. Therefore, HA may act directly on the tumor cell or on a component of the microenvironment.

In tumor tissue, an increased level of HA synthesis is noted. When fibroblasts and breast cancer cells were co-cultivated, an increase in the expression of the HAS2 enzyme was noted on fibroblasts, which in turn increased the amount of synthesized HA. In this case, a certain protein synthesized by the cells of the tumor itself, С10orf118, is indicated as a stimulus for HAS2 hyperexpression [30]. However, this is not the only example of the factors through which tumor cells are able to increase HA synthesis by fibroblasts. HAS2 expression is also regulated by interleukin-1β, FGF-2, PDGF, KGF, EGF, and TGF-β in tumor-associated fibroblasts [31,32].

HA synthesized by tumor-associated fibroblasts themselves, has powerful autocrine and paracrine effects in ensuring the migration of both tumor-associated fibroblasts and tumor cells themselves. Hyaluronic acid binds to cell receptors, which additionally promotes the migration and invasion of tumor cells following tumor-associated fibroblasts. [33] The role of HA and its receptors in the motility of tumor cells and their migration has been demonstrated using colorectal cancer and pleural mesothelioma as examples [34,35].

Also, HA in the stroma of solid tumors stimulates the development of PD-L1-expressing macrophages, which reduces the T-cell immune response (via the PD1/PD-L1 pathway) and forms an immunosuppressive microenvironment [36].

4. Hyaluronic Acid and СD44

CD44 is a type I non-kinase transmembrane protein involved in cell adhesion, migration, and cell-cell interactions [37]. Its role in various biological processes is known, including hematopoiesis, embryogenesis, inflammation, and regeneration. The CD44 gene is located on the short arm of chromosome 11 in humans and on chromosome 2 in mice and has several exons that can undergo alternative splicing, resulting in the formation of variant isoforms of this receptor. Dozens of different CD44 isoforms have been described to date. Among them are the standard isoform (CD44s), the epithelial form (CD44E), and numerous variant isoforms (CD44v) [38,39]. CD44 is expressed ubiquitously in the body, but CD44 expression at the isoform level can vary depending on the tissue type (Figure 2) [40,41,42]. CD44 can bind to various ligands, such as osteopontin, chondroitin, collagen, fibronectin, laminin, serglycin, but the main ligand for CD44 is HA [39]. The binding activity of CD44 with various ligands, and therefore its functionality, largely depend on the CD44 isoform and its conformational features [38].

To date, a large amount of data has been accumulated indicating the participation of CD44 isoforms in the regulation of signaling pathways (MAPK, Hippo, Hedgehog, PI3K/AKT/mTOR, Twist, HIF, c-Src, Wnt) responsible for: progression of various tumors, epithelial-mesenchymal transition, invasion and metastasis, as well as resistance to antitumor therapy [43,44,45]. Moreover, many studies demonstrate a correlation between CD44 overexpression and poor prognosis. Thus, the contribution of CD44 to the development of the tumor process, through the above mechanisms, has been described for brain cancer, head and neck cancer, breast cancer, kidney cancer, liver cancer, pancreatic cancer, gallbladder cancer, esophageal cancer, prostate cancer, gastrointestinal cancer, melanoma, squamous cell carcinoma, sarcoma. A similar role for CD44 has been described for hematological malignancies such as acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphoblastic leukemia, chronic myeloid leukemia, and multiple myeloma.

The expression of CD44 isoforms and their expression level often vary depending on the tumor type. Tumor cells can express a different repertoire of isoforms simultaneously, although, as a rule, a predominant isoform stands out among them. For example, in patients with acute myeloid leukemia and glioblastoma, the expression of CD44s prevails, while in patients with colorectal cancer, the expression of CD44v8-v10 prevails in more than 75% of cases [46,47]. Moreover, according to some data, the expression of CD44 isoforms can be plastic depending on the tumor stage. Thus, some studies demonstrate that, on the one hand, CD44 expression is necessary for tumor initiation and metastatic activity, and on the other hand, suppression of CD44 expression may be beneficial for tumor progression [40]. Such plasticity can be explained by the fact that oncogenic signals are involved in the regulation of variant CD44 exons [48]. As for the specific contribution of different isoforms to tumor development, in most cases, CD44s expression is associated with tumor growth, while CD44v isoforms are more associated with invasiveness and chemoresistance [43].

CD44 actively participates in the process of tumor metastasis. This effect may be due to a parallel with the physiological migration of immature or activated lymphocytes expressing multiple variants of CD44 isoforms [49,50,51,52]. Interestingly, the ability to metastasize can be provided by both CD44s and various variants of CD44v [53,54,55,56,57,58]. Moreover, each tumor type apparently has its own pattern of CD44 expression responsible for migration. For example, non-metastatic rat pancreatic cancer cells transfected with CD44v acquired the ability to metastasize. CD44v6 has also been shown to be responsible for lymph node metastasis in intraductal breast carcinoma. In turn, CD44v6 expression by cancer stem cells (CSC) initiated the metastatic process in colorectal cancer. On the other hand, CD44s has been demonstrated to be responsible for metastatic activity in human lymphoma. However, other studies have demonstrated that CD44 expression is dispensable for tumor metastasis. Deletion of CD44 alleles did not limit in vivo metastatic capacity for MDAY-D2 lymphosarcoma. In contrast, lack of CD44 in mice impaired osteosarcoma metastasis.

CD44, individually or together with other molecules (CD24, CD133, CD34 and c-Met) can be a key marker for cancer stem cells [59]. Thus, for a number of solid tumors CD44 binding to ECM to the maintenance of the stemness of malignant cells [60]. Moreover, intact nuclear CD44/STAT3 signaling has been shown to be critical for reprogramming cancer cells to a CSC phenotype through transcriptional regulation of c-myc expression and subsequent self-renewal of CSCs [61]. Interestingly, CSCs may have a unique expression pattern of CD44 isoforms depending on the tumor type. For example, CD44v6 is characteristic of CSCs in colorectal cancer [62], CD44v8-10 in gastric cancer [63], and CD44v3 in head and neck cancer [64]. It is assumed that CSCs are responsible for tumor heterogeneity, which is the main obstacle to the success of antitumor therapy [65]. Taken together, multiple studies in the literature suggest that CD44 is expressed on tumor cells, including cancer stem cells, and is a promising target for targeted therapy in oncology.

5. Hyaluronic Acid and Other Receptors of HA

The receptor for hyaluronic acid-mediated motility (RHAMM) is localized inside the cell and is unconventionally exported to the cell surface in response to certain defined stimuli such as wounding [66]. The mRNA of RHAMM is present in many tissues and cells, most abundantly in cells of the immune system, gastrointestinal tract, reproductive system and in tumor [41]. There is a direct correlation between the level of RHAMM expression and worsening prognosis in various types of cancer [67,68,69]. This can be explained by increased cell migration of tumor cells upon interaction with the RHAMM receptor of low-molecular HA [70,71,72,73]. However, opposite results were also obtained when genomic reduction of RHAMM led to the emergence and development of malignant tumors of the peripheral nerve sheaths [74]. Such ambiguous results can be explained by the ability of RHAMM to bind to numerous proteins and polysaccharides, its multifunctional nature. The LYVE1 receptor is located not only on the endothelial cells of the lymphatic vessels, but also on a certain subpopulation of macrophages (LYVE1⁺ macrophages). It is known that LYVE1⁺ macrophages are involved in the internalization and degradation of HA. It was shown that upon depletion of LYVE1⁺ macrophages, there was an excessive accumulation of HA, which had suppressive properties with respect to tumor cells [75]. The possible role of excessive amounts of high-molecular weight hyaluronic acid in antitumor protection is also discussed using naked mole rats as an example in the work of Xiao Tian et al. [76]. Upon suppression of HAS2 expression or hyperexpression of enzymes that break down HA, it was shown that naked mole-rat cells became more vulnerable to malignancy. The authors found that high-molecular weight hyaluronic acid plays an important role in the activation of the mechanism of early contact inhibition of cells. Activation of early contact inhibition of cells is explained by the inclusion of the HA/CD44/NF2 pathway.

6. HA-Based Drug Delivery Systems for Solid Tumors

In the case of HA-based delivery systems, hyaluronic acid, which participates in the construction of the system, can serve as a ligand [77,78,79,80]. Hyaluronic acid can bind to various receptors on the cell surface, including CD44, which is expressed at a high level on tumor cells. A conjugate of hyaluronic acid and paclitaxel (Oncofid-P) is currently undergoing clinical trials as a drug for the treatment of bladder carcinoma and mesothelioma. In addition, HA can be chemically modified to enable the development of advanced drug delivery systems and biomaterials with enhanced functionalities and targeted applications [81].

The combination of HA and other molecules included in the drug delivery system not only maintains the ability of hyaluronic acid to target a specific target, but also provides the system with the ability to deliver chemotherapeutic drugs through multiple interactions with tumor microenvironment components of solid tumors. Supramolecular delivery systems based on a hyaluronic acid framework and a macrocycle are one of the promising directions for drug delivery [82,83]. There are various other delivery systems based on hyaluronic acid, including nanoparticles, nanogels and niosomes [84,85,86,87,88,89]. Additional molecules in the drug delivery system may enhance targeting and/or improve the antitumor properties of the drug. For example, аntitumor effect in vitro and in vivo in a mouse model was obtained using HA nanoparticles with a bilirubin core loaded with doxorubicin. The HA shell allowed the particle to target the CD44 overexpressing cell, and bilirubin enhanced the antitumor effect of the drug and its release in a ROS-containing environment [90].

One of the drawbacks of hyaluronic acid-based drug delivery systems is their poor accumulation in solid tumors due to the superficial penetration depth, low cellular uptake, and non-specific drug release [91]. As described above, hyaluronic acid receptors, including CD44, are widely present in the body and are found not only on tumor cells. Moreover, high expression levels of CD44 on blood cells hinder the transport of the drug delivery system in the bloodstream. Therefore, various methods are often used to enhance targeted delivery, such as dual targeting [92]. Li et al. designed a HA-based drug delivery system that released an immune regulator when located in an immune cell and released doxorubicin in the case of a tumor cell with a lower pH [93]. Zhang et al. [91] prepared a HA nanoparticle conjugated with both NO prodrug (alkynyl-JSK) and doxorubicin prodrug (cis-DOX) for breast cancer therapy. This nanoparticle achieved cascade-enhanced drug delivery efficiency based on a relay strategy of deep tumor penetration, NO uptake by CD44 tumor cells, and HA release of tumor microenvironment-sensitive drug.

7. HA-Based Drug Delivery Systems for Hematological Malignancies

Hematological malignancies are formed from hematopoietic or lymphatic tissues. To maintain therapeutic levels in the bone marrow or lymphatic system, chemotherapeutic drugs require high doses or more frequent administration, which can lead to increased side effects [94,95]. In addition, the bone marrow microenvironment may contain a huge number of tumor stem cells that are resistant to chemotherapy and mediate refractoriness to therapy and disease relapse [96,97].

According to the literature, the expression of CD44, a receptor for hyaluronic acid, has been found in many hematological malignancies [98]. CD44 is involved in the development and progression of hematological malignancies by enhancing apoptotic resistance, invasiveness, as well as regulating bone marrow homing and mobilization of leukemia-initiating cells into the peripheral blood. Also, CD44 is prognostic marker indicating an increased likelihood of disease relapse [99]. Elevated CD44 expression may serve as a marker of worse prognosis in most hematological malignancies Expression of CD44 on tumor cells in B-cell acute lymphoblastic leukemia is a risk of relapse after elimination of the primary lesion [100,101]. In acute T-cell lymphoblastic leukemia, high levels of CD44 on cells were noted, and a positive correlation was observed with the development of infiltration of the internal organs of patients [102]. The role of the NOTCH1-MYC-CD44 axis in the risk of relapse in T-cell acute lymphoblastic leukemia associated with the persistence of leukemia-initiating cells was also noted [103].

Despite numerous data on the role of CD44 in tumors of hematopoietic and lymphoid tissues, CD44 has been rarely considered as a target for targeted therapy for this type of pathology [104]. Thus, nanoparticles were created for the treatment of non-Hodgkin’s lymphomas, the outermost layer of the particle consisted of hyaluronic acid, covalently conjugated with antibodies to CD20, which provided dual targeting to tumor cells [105]. To provide antitumor action, the nanoparticles were loaded with siRNA. It was found that the introduction of the HA-based drug delivery system induced apoptosis of target cells and prevented the proliferation of blood cancer cells both in cell culture and in animal models of non-Hodgkin’s lymphoma. A similar approach was used in a similar system, only the authors performed triple targeting (hyaluronic acid and two antibodies to surface markers) [106]. A different approach was used by another group of authors, who took doxorubicin-loaded nanoparticles as a basis [107]. The particle surface was modified with hyaluronic acid for targeted delivery. It was shown that doxorubicin encapsulated in hyaluronic acid nanoparticles cross-linked with lipoic acid (LACHA-DOX) mediated highly effective and targeted inhibition of human tumor cells, including human multiple myeloma LP-1 and human acute myeloid leukemia AML-2, during xenotransplantation of cells into nude mice. Duvelisib loaded nanoparticles coated with hyaluronic acid have demonstrated their safety and efficacy in models based on CD44-positive cell lines of hematological malignancies [108]. Silver-based nanoparticles coated with hyaluronic acid enhanced ROS production in leukemia cells and induced tumor cell apoptosis [109]. Therefore, the results indicate the effectiveness of targeting HA-based delivery systems for the treatment of blood cancers.

8. Conclusions

HA has optimal properties such as low toxicity, high biocompatibility, biodegradability, and stability which allows it to be widely used in the creation of drug delivery systems. Many tumors, including various solid and blood cancers, highly express receptors for hyaluronic acid. As the present studies indicated, HA-based drug delivery systems have shown their effectiveness and safety. However, the use of targeted therapy on HA-based delivery systems does not always allow achieving the desired therapeutic effect due to low accumulation in the tumor. Moreover, in most cases, the development of new HA-based delivery systems is directed towards the treatment of solid tumors, while hematological malignances often remain without due attention, despite the high expression of CD44 on blood cancer cells. Therefore, more advanced and multi-component drug delivery systems are needed to eradicate various types of cancer with greater efficacy, selectivity and less toxicity, confirmed by clinical trials.