Targeted therapeutic effects of oral inulin-modified double-layered nanoparticles containing chemotherapeutics on orthotopic colon cancer
Introduction
Colon cancer is one of the most common cancers globally. Global Cancer Statistics of 2020 reported 1.93 million new cases and 940,000 deaths related to colorectal cancer worldwide, making it the third most common cancer and the second in cancer mortality worldwide [1]. Although chemotherapy is widely used and important treatment option for colon cancer, emerging evidence shows that the systemic circulation of chemotherapy drugs attacks both cancer cells and normal cells, which cause great trauma and toxic side effects to patients. In addition, chemotherapy drugs are easily inactivated in the systemic circulation, therefore, do not achieve the best therapeutic effect [[2], [3], [4]].
Oral colon-targeted drug delivery systems allow for greater dosage flexibility and self-administration and contribute to greater patient compliance [5]. However, limitations, including poor absorption of oral chemotherapy drugs, instability in the gastrointestinal tract (GIT), rapid degradation, and inactivation during internal circulation pose challenges in ensuring the selective increase of drug concentration in the lesion [6]. Moreover, the short residence time of the drug at the tumor site does not favor the selective increase of drug concentration in the colon cells [5,[7], [8], [9]]. Nano-drug delivery systems have greater potential to target specific organs, increase cycles, and control systemic release; as such, they allow sufficient drug accumulation in the cytoplasm to improve chemotherapy outcomes significantly [10]. Of note, these unique properties of nano-drug delivery systems offer promising strategies for overcoming the barriers to colon cancer treatment and achieving maximum efficacy in colon tissue [11,12]. Compelling evidence indicates the potential of nano systems for oral administration of colon cancer at the preclinical level [[13], [14], [15]].
In the GIT, a series of physical, chemical, and enzymatic barriers hinder oral drug delivery and stability and are considered among the major problems to be overcome [16]. Research shows that transport time and a large number of enzymes in the human GIT, including pepsin, trypsin, and glucosidase are major hindrance factors of colon-specific delivery [9]. Enzymes in the upper GIT digest or degrade the orally administered drugs, hampering the complete delivery of the drugs to the colon tumor site. In addition, mucus, a gelatinous layer covering the intestinal epithelium, can protect the epithelial cell surface by trapping and eliminating pathogens and foreign particles, thereby limiting the therapeutic effect of drugs in the colon [17,18]. To overcome these obstacles, there is a need to design safe and efficient oral colon-specific drug delivery systems to ensure the safe delivery of drugs to the colon and the cross-mucosal ability of drugs in the colon.
In the current research, several strategies for the colon system based on natural polysaccharides have shown great potential to reach the colon region, which allows for greater colon-targeting specificity and increased drug accumulation/concentration at the disease site [5]. Inulin (IN), which is approved by the American Food and Drug Administration (FDA) for intravenous and oral administration, is a cheap, safe, and biodegradable natural polysaccharide extracted from plants [[19], [20], [21]]. IN has become the most popular colon-targeting carrier due to its unique properties, including protection of drugs from the acidic environment of the stomach and upper digestive tract and ensuring inulinase degradation in the colon to allow controlled drug release [[22], [23], [24], [25], [26], [27]]. More evidence shows that being a king of short-chain fatty acids produced via colonic bacteria-triggered fermentation degradation, IN can exert potential probiotic effects on gut [[28], [29], [30], [31], [32], [33]]. Hyaluronic acid (HA) is a biocompatible and degradable natural anionic material that potentially promotes the passage of nanoparticles through the mucus layer following modifications on the surface [17,18]. HA also serves as a tumor marker that targets the overexpressed CD44 receptor in tumor tissues to ensure CD44-mediated uptake in tumor cells [[34], [35], [36], [37], [38]]. Studies have demonstrated that HA modified nanoparticles can realize both the transmembrane effect of drugs in colon mucus and the active targeting effect and elevate drug accumulation in tumor tissues [12,37,39].
In the present study, the polymer PLA-PEI and PTX formed stable PTX/PLA-PEI nanoparticles (PTX/PP NPs) by self-assembly. Then, the negatively charged HA terminal in HA-IN was integrated with the positively charged PEI of PLA-PEI to get our final composite nanoparticles of PTX/PLA-PEI/HA-IN (PTX/PPHI NPs, Fig. 1A). The incorporated PTX could not be degraded in the upper digestive tract because of the IN outer, and thus solved the first obstacle of oral administration in the GIT. After reach the lower digestive track, owing to the negative charge characteristics of the mucus layer, negatively charged PTX/PPHI NPs potentially enhance mucosal fluidity through charge repulsion. This promotes the rapid diffusion of PTX/PPHI NPs in the mucus layer [17,18]. At this point, the IN in the outermost layer of composite nanoparticles is degraded by the colon-specific enzymes, and the exposed HA could attribute to improve the permeability and trans-mucosal transportability [18]. A proportion of nanoparticles may enter the blood circulation thereby passively and/or actively targeting the colon tumor cells under the specific “EPR effect” and/or the HA targeting [40]. The rest of the nanoparticles trapped in the colon attempts to cross the colon mucosa and enter the colon tumor site with the help of HA. Furthermore, PEI-modified composite nanoparticles can cause lysosome swelling and rupture, known as the “proton sponge” effect [41,42], thereby releasing drugs into the cytoplasm and exerting an anti-tumor effect (Fig. 1B). This microbiome responsive targeting of nanoparticles holds great promise as a potential approach for effective orthotopic colon cancer management.
Section snippets
Materials
Poly (lactic acid) with carboxyl groups on one end (PLA-COOH, MW = 2.5 kDa) was supplied by Jinan Daigang Technology Co. (Shandong, China). Branched polyethyleneimine (bPEI1 k, MW = 1 kDa) was purchased from HWRK Chem Co., LTD. (Beijing, China). Inulin (MW ≈ 5 kDa), Inulinase, Chlorpromazine, Methyl-β-cyclodextrin, Amiloride Hydrochloride, Chloroquine and Wheat Germ Agglutinin (WGA) were purchased from Sigma-Aldrich (Shanghai, China). 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
Synthesis and characterization of PLA-PEI and HA-IN copolymers
The products were characterized by 1H NMR and FTIR. Amphiphilic PLA-PEI polymer was synthesized via the reaction between the –COOH group of PLA and the –NH2 group of PEI in the presence of EDC/NHS. 1H NMR of synthesized PLA-PEI showed a series of wide peaks at δ = 2.87 ppm, belonging to PEI's -CH2CH2- proton absorption peak. The two new absorption peaks at δ = 1.71 ppm and δ = 4.78 ppm were derived from the –CH3 and –CH proton absorption peaks of PLA, respectively (Fig. 2B). In FTIR of PLA-COOH
Conclusion
In this current study, we developed an oral colon dual-targeted composite nanoparticle loaded with chemotherapy drug PTX. Our data showed that the PTX/PPHI NPs had suitable in vitro stability in simulated gastrointestinal environments and efficient colon targeting ability. The IN of the outer layer can protect drugs from the acidic environment of the stomach and upper digestive tract but could be degraded by the colonic enzymes. Upon the enzymatic degradation and removal of the “IN shells”, the
Credit author statement
Yan Hou: Conceptualization, Methodology, Software. Jingzhe Jin: Investigation, Writing – original draft. Hongxia Duan: Writing – original draft, Resources. Chao Liu: Software, Validation. Liqing Chen: Writing- Reviewing and Editing. Wei Huang: Supervision, Resources. Mingji Jin: Validation, Funding acquisition. Zhonggao Gao: Validation, Funding acquisition.
Declaration of competing interest
The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Mingji Jin reports financial support was provided by National Natural Science Foundation of China. Zhonggao Gao reports financial support was provided by National Natural Science Foundation of China. Liqing Chen reports financial support was provided by Fundamental Research Funds for the Central Universities.
Acknowledgements
We acknowledge the support from the National Natural Science Foundation of China ((82104106, 82073778) and the Fundamental Research Foundation for the Central Universities (3332021044, China).
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