Polymeric micelle/nano hydrogel composite matrix as a novel multi-drug carrier

Highlights

• Novel multi drug delivery system having heparin as the back bone.

• Controlled and targeted delivery in colorectal cancer.

• Combined effect of cisplatin and curcumin by MTT assay.

• Micellar DDDS having anticoagulant, inhibition of angiogenesis and antitumor activity.

Abstract

According to the World Health Organization (WHO), fatality of cancer is 9.6 million in 2018. Of these, 1.8 million deaths are due to colorectal cancer. However, it doesn't seem that proper medications are unavailable. Instead, the medications should reach the site of action without causing any further side effects. With the advent of smart biomaterials this target is on its verge to be achieved. Previous reports prove that the side effects induced by Cisplatin (CIS) could be greatly decreased by curcumin (CUR). Hence, if these two drugs are used in unison, greater efficacy with lesser toxicity could be achieved. However, to incorporate two drugs and to release them simultaneously without any premature leakage can be achieved by polymeric micelles. To increase the core and hydrophobicity, monomers with large alkyl chains like triethylene tetraamine (TETA) and oleic acid (OA) were grafted to the backbone of heparin (HEP) skeleton. To target these drugs, folic acid was entrapped within hydroxyl appetite (HAP) based hydrogel and was dispersed onto polymeric micelle (PM). This coating could further act as a potential barrier to sustain the drug release as evident from similar previous studies. All the reaction steps were monitored using FTIR, XRD, H¹ NMR, DLS and SEM studies. The in vitro swelling and release studies proved promising with folic acid showing burst release followed by the sustained release of chemotherapeutic drugs. The efficacy was further ascertained by MTT assay on normal and HCT 116 colon cancer cell lines. This was further proved by fluorescence studies. Thus, the present material could act as a promising candidate for the treatment of colorectal cancer with high efficacy and minimal toxicity.

Introduction

Colorectal cancer is the third most common type of cancer in humans [1,2]. Patients in the advanced stage suffer severe physiological changes and require intensive chemotherapy that often results in poor response and significant morbidity. The undesirable effects of intensive chemotherapy on normal cell and the development of multidrug resistance are the main cause of treatment failure. Recent advances in DDS, makes it possible to target cancer cell line using cytotoxic drugs without affecting normal cells. Nano carriers such as micelles, hydrogels, polymeric nanoparticles and liposomes are capable to improve drug distribution and bioavailability with enhanced cytotoxic concentration in the tumor mass with simultaneous drug delivery leading to reduced side effects [3,4].

Biopolymers are widely being used as a raw material for the design of drug delivery formulations because of their outstanding properties such as biocompatibility, non-toxicity, biodegradability and environmental sensitivity [5,6]. Most common biopolymers include chitosan, sodium alginate (SA), guar gum and heparin (HEP) [7,8] and are being widely used for controlled drug delivery applications. However, the flaws, including weak mechanical properties and burst release of drugs is inevitable when pristine biopolymers are used as carriers. The anticoagulant activity of HEP is greatly reduced after the formation of nano particles and its nano particles do not induce significant toxicity after intravenous injection. Hence, for unconventional routes of drug delivery, HEP seems to be a potential precursor [9].

Hydroxyapatite (HAP, Ca₁₀ (PO₄)₆ (OH)₂) is another non-viable and most important bio mineral found in naturally hard tissue. Because of its biocompatibility, bioactivity, nontoxicity, osteo conductivity, non-inflammatory and non-immunogenic properties, it is widely used for biomedical applications [10,11]. HAP nano composite porous scaffolds were fabricated biometrically and their feasibility as a drug delivery carrier for tissue regeneration and wound healing treatment were evaluated previously [12,13].

Micellar carrier systems are investigated to improve drug solubility and stability. Due to toxic and immunogenicity, natural polysaccharides act as substitutes for synthetic polymers in the development of new micelle systems. By grafting hydrophobic moieties to the polysaccharides backbone, self-assembled micelle can be readily formed in aqueous solutions [14,15]. HEP based micelle systems possess wide variety of biological functions such as anticoagulant activity, inhibition of angiogenesis and antitumor development [16]. The over expression of folate receptors in tumor sites is used as a tool for targeting chemo therapeutic drugs. The folate receptor situated in the surface of the cancer cell can combine with FA or its conjugates to form endosomes [17,18]. These conjugated molecules can be further coated with poly ethylene glycol (PEG) to target the folate binding proteins. For conjugating with FA, long chain fatty acids like hexanoic acid, linoleic acid, stearic acid and oleic acid were used in previous studies [19,20]. Up to date, different hydrophobic molecules, such as octaldehyde [15], Palmitic acid and Stearic acid have been grafted with chitosan to get amphiphilic derivatives, so as to form micelles or micelle like self-aggregates as drug carriers.

At low temperature, a thermo reversible and biodegradable polymer in aqueous solutions is in solution mode and becomes a gel at body temperature. This unique property gives hydrogels more significance in drug delivery as drugs, proteins and cells can be easily mixed with polymeric aqueous solutions at low temperature and can be trapped in the gel state upon in vivo administration [20], [21], [22]. The development of hydrogels in polyesters has made great attention, because the copolymers of polylactide (PLA), polyglycolide (PGA) and polyethylene glycol (PEG) have been approved by FDA for in vivo use [23].

Combinatorial use of multiple drugs has immense applications in pharmaceutical field, getting a better outcome than mono chemotherapy [24]. Multi drug delivery involving two or more drugs is emerging as a challenging approach for the treatment of cancer. This combinational therapy approach could significantly reduce multi drug resistance and side effects leading to enhanced therapeutic efficiency. CUR (diferulolylmethane), is a yellow pigment which exhibit antioxidant, anti-inflammatory and anti-cancer properties and has been described as a neuro protector against neurological disorders [25], [26], [27]. Cisplatin (CIS) is another organometallic drug with excellent chemotherapeutic index. Due to its activity, low amounts of drug are administered thereby decreasing the dose. However, neurite growth is a serious side effect of CIS administration owing to the heavy metal intake [28,29]. It is in this scenario that combinatorial approach where, drugs act in unison comes to the fore. As per previous reports, when CUR is used along with CIS, the side effect of CIS is found to be greatly reduced as evident from the in vitro investigation of chemotherapy induced peripheral neuropathy. Derivatives of CUR when used in unison with CIS also proved to be beneficial for simultaneous intracellular delivery to lung cancer [30].

Combinatorial therapy will enhance the drug usage than conventional methods. Since most of the polymer drug nano composite that has been explored were designed for the anticancer therapy [31,32]. Due to extensive chemo therapeutic procedures, it is very normal for a cancer patient to lose bone density and is often prescribed with hydroxyl appetite drugs or calcium supplements. It was in this direction, HAP was selected as the drug carrier. To target the chemotherapeutic agents to tumor sites, FA was hydrogen bonded with HAP hydrogel.

In the present work, all these aspects were severely scrutinized with the aim of developing a highly patient compliant DDS. In the present approach, two drugs were used in unison – CUR and CIS, not only to increase the overall efficiency of the DDS, but also to decrease the side effect of one drug by the other. To ensure better drug encapsulation of dual drugs and to avoid premature leakage, they were entrapped within the hydrophobic core of the PM composed of HEP. The core of the micellar structure was ensured to be large and hydrophobic enough to accommodate simultaneously CIS and CUR by grafting HEP with triethyl tetraamine (TETA) and oleic acid (OA). To sustain the release of these drugs, the hydrophilic tail of the micelle was coated with a hydrogel of HAP, preloaded with FA. Even though, our group itself has reported the combination of micelle/hydrogel composites, the possibility of improving the void space of the micelle to prepare a carrier for multiple cargo is evaluated for the first time. All steps were carefully monitored using FT-IR, XRD, DLS and SEM analysis. Swelling studies were conducted at different pH level which revealed the swelling dependency of the material in drug release. The cytotoxicity study was performed in colon cancer cell line - HCT116 and results were comparable with the previous literature. To conclude, pH and body temperature favors the release of drugs and it shows toxic effect only in the colon cancer cell lines without affecting the normal cell line. Hence, the prepared material serves to be a promising candidate for the upcoming drug delivery technology.