Synthesis of a smart pH sensitive micelle containing hyaluronic acid-curcumin bioconjugate against colorectal cancer

Synthesis of a smart pH sensitive micelle containing hyaluronic acid-curcumin bioconjugate against colorectal cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Synthesis of a smart pH sensitive micelle containing hyaluronic acid-curcumin bioconjugate against colorectal cancer Niloufar Hazrati, Sadegh Dehghani, Sahar Taghavi, Seyed Mohammad Taghdisi, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4216826/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Nov, 2024 Read the published version in BioNanoScience → Version 1 posted 9 You are reading this latest preprint version Abstract In the current study, we fabricated a pH-sensitive self-assembled CD44-targeted therapeutic micelle, comprising curcumin (CUR)-hyaluronic acid (HA) conjugate. At the first stage, the biopolymer, HA, as a back bone was attached to ethylene glycol vinyl ether (equivalent to 50% of the carboxylic acids of HA) and then hydroxyl of curcumin was attached to this linker to form a pH-responsive acetal linkage. The prepared HA-CUR conjugate was self-assembled and formed a micellar structure with size of 84 nm. The release of CUR from the prepared platform illustrated a controlled, sustained release at pH 7.4 while it was significantly accelerated at pH 5.4. The cytotoxicity and cellular uptake of the platform were evaluated against C26 as a CD44 positive and CHO as CD44 negative cells. The cytotoxicity and cellular uptake study showed higher internalization and cellular toxicity of the synthesized platform in C26 cells compared with CHO cells. In vivo study demonstrated desirable therapeutic efficacy of HA-CUR toward C26 tumor growth suppression and survival rate of BALB/c mice. These findings suggested HA-CUR as a hopeful natural product-based nanomedicine for active targeting and delivery of CUR to colon adenocarcinoma. Curcumin Colorectal cancer Hyaluronic acid Targeted drug delivery Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Colorectal cancer (CRC) is known as one of the deadliest cancer in both genders, worldwide. In 2023, approximately 153,020 new cases have recognized with CRC and this disease caused the death of 52,520 individuals [ 1 , 2 ]. Different conventional treatment options have been implemented for CRC such as chemotherapy, radiation therapy, and surgery, but these treatments showed undesirable adverse effects which can significantly affect patients' quality of life [ 3 , 4 ]. Additionally, cancer cells can develop resistance to chemotherapy and radiation, causing the failure of chemo and radio-therapy [ 5 , 6 ]. As a therapeutic strategy to improve cancer treatment outcomes, scientists have been exploring alternative therapeutics, including natural compounds comprising curcumin, resveratrol, lycopene and gingerol. These compounds have been found to have chemopreventive and/or anticancer properties with minimal side effects [ 7 ]. In this regard, curcumin as a natural polyphenol and the active component of a medical plant named Curcuma longa has shown promising antimicrobial, anti-inflammatory and wound healing activities [ 8 ]. Furthermore, numerous in vitro and in vivo experiments reported anti-tumor characteristics for curcumin against lung cancer, pancreatic cancer, brain tumors, cervical cancer, and colorectal cancer [ 9 – 12 ]. However, low aqueous solubility, poor bioavailability, rapid metabolism and chemical instability of curcumin have restricted its therapeutic efficacy [ 13 ]. Over the past years, various nanotechnology-based drug delivery systems were designed to improve curcumin pharmacokinetics and overcome the aforementioned limitations toward systemic delivery. These nanoparticulate systems such as nanoemulsion, solid lipid nanoparticles, phytosomes, gold nanoparticles, liposomes, and polymersomes improved therapeutic index of curcumin [ 11 , 14 ]. Among different nanoplatforms, polymer-drug conjugates or therapeutic polymers are promising platforms which could increase solubility and stability of curcumin while improving its pharmacokinetics. In these systems, stimuli-sensitive linkage between polymer and drug is formed which responds to inherent or external environmental stimuli such as reactive oxygen species, light, ultrasound, temperature, specific enzymes, and pH, thereby providing an intelligent smart platform [ 15 – 18 ]. Inserting of acetal linkage as pH-sensitive bond between polymer back bone and drug can control and allow triggered release of chemotherapeutic agent from the conjugate at the intended site, under acidic environment in lysosomes and tumor tissues, enhancing the therapeutic efficiency and diminishing chemotherapy related side effects [ 9 , 10 , 19 ]. HA is known as a biodegradable natural polymer with a linear structure consisted of alternating repeats of N-acetyl-D-glucosamine and D-glucuronic acid [ 20 ]. HA can be chemically modified through its carboxylic acid and hydroxyl groups [ 21 , 22 ]. HA-drug conjugates are suitable drug delivery platforms for different therapeutic agents due to the biocompatibility, nontoxicity, nonimmunogenicity and targeting capability of HA to CD44 receptor which is overexpressed in a majority of cancerous cells [ 23 – 26 ]. Moreover, HA is degraded by hyaluronidases, such as HYAL1, resulting in low molecular weight components after internalization into cancer cells through CD44 receptor-mediated endocytosis [ 27 , 28 ]. The current work reports on the synthesis of a novel multifunctional nanoplatform derived from HA conjugated with curcumin (HA-CUR) through pH-sensitive acetal linkage for on-demand release and active targeting of CD44 receptors against colorectal cancer. The current study investigated the hypothesis that this therapeutic pH responsive polymer-drug conjugate could self-assemble to micellar structure and the hyaluronic acid as a targeting ligand on the micellar surface could enhance the cellular uptake and anti-cancer potency of synthesized HA-CUR nanoplatform through CD44-mediated endocytosis. Various experiments were conducted to characterize the structure, size, release kinetics, anticancer activity, and intracellular entrance of the prepared nanoplatform. Moreover, biodistribution and the therapeutic index of HA-CUR nanoplatform were investigated in vivo on subcutaneous C26 murine colon cancer model. 2. Materials and methods 2.1. Materials See supporting information for details. 2.2. HA-CUR conjugate synthesis 2.2.1. Characterization In each step of the synthesis procedure, the product in deuterated solvents were characterized by 1 HNMR (proton nuclear magnetic resonance) spectroscopy using 1 HNMR spectrometer (Bruker, Rheinstetten, Germany). The Perkin Elmer FTIR spectrometer was used to obtain Infrared spectra (FTIR) of HA, CUR and HA-CUR conjugate in KBr pellets. 2.2.2. Sodium removal from sodium hyaluronate For sodium removal from sodium hyaluronate, sodium hyaluronate (500 mg) was dissolved in 10 mL of HCl (0.1 N), followed by stirring for 3h at room temperature. Afterwards, it was placed into a dialysis tube (M W cut off = 2 kD) overnight against one liter of deionized water at 4 ◦ C. Ultimately, the white powder of purified sodium-free HA was obtained by freeze-drying and stored at − 20°C. 2.2.3. Conjugation of HA to EGVE In the next step, sodium-free HA was conjugated to the ethylene glycol vinyl ether (EGVE) linker (equivalent to 50% of carboxylic acid groups of HA). For this purpose, 100 mg of sodium-free HA was dissolved in 10 mL anhydrous DMSO, then DMAP was added (molar ratio of EGVE to DMAP was 1:4) to the mixture and stirred for 10 min. Next, DCC was added (molar ration of DMAP:DCC was 1:1) and further stirred for 2 h to make active carboxylic acids of HA. Then, EGVE was added and gently stirred overnight at 50°C. In the next step, under vacuum the solvent was removed and the suspension of HA-EGVE was poured into dialysis tubing with a cut off of 2 kD in order to dialyzed against distilled water for 24 h. Finally, the HA-EGVE was lyophilized and stored at -20°C. 2.2.4 Conjugation of curcumin to the HA-EGVE For fabrication of pH-sensitive acetal bond between EGVE and CUR, 6 mL of DMSO was added to HA-EGVE (100 mg) and sonicated for 30 min at 60°C. Next, in order to functionalize the synthesized HA-EGVE, p -toluenesulfonic acid monohydrate and CUR (molar ratio of p -toluenesulfonic acid monohydrate: EGVE:CUR was 1.5:1:1) were added and stirred overnight. The resultant product was dialyzed using dialysis tube with 2 kDa cut off against distilled water overnight. Then, the HA-CUR was lyophilized and kept at -20°C. All synthesis steps were conducted under dark conditions to avoid CUR degradation [ 29 ]. 2.3. Self-assembly of HA-CUR HA-CUR conjugate was self-assembled through direct hydration technique. For this purpose, one mg of HA-CUR conjugate was dispersed in distilled water and bath sonicated for 15 min. The content of the conjugated curcumin was calculated, implementing the UV absorbance of CUR at 420 nm (Varian CARY 100 spectrophotometer, California, USA). To this aim, 1 mL of DMSO was added to 1 mg of HA-CUR and the UV absorption of the solution was measured at 420 nm. Then, the CUR content was calculated using standard curve of CUR. 2.4. Morphological characteristics To investigate the morphological features and size of the HA-CUR NPs in deionized water (100 µg/1mL), with field emission scanning electron microscope (FESEM), using TESCAN BRNO- Mira3 LMU instrument made in Czech Republic and dynamic Light-Scattering (DLS) Instrument-Malvern Instruments Zetasizer NanoSampler (Malvern, United Kingdom). To this aim, HA-CUR NPs (1 mg/mL) were poured on an aluminum stubs, dehydrated, and then covered with a thin layer of gold to create the connection with the aluminum surface. Finally, the imaging procedure was operated at 10 kV. 2.5. CUR release profile For investigation of CUR in vitro release from HA-CUR NPs, dialysis method was implemented. In this regard, 1 mL of HA-CUR suspension was poured into two dialysis sacs with M W cut off = 2 kD, immersed in either 25 mL of PBS (pH 7.4) or citrate buffer (pH 5.5), supplemented with 0.5% tween 80 and maintained in a shaker incubator (37°C, 80 rpm). The release media were protected from light during the experiment. At predesigned times, 1 mL of each vessel was taken out and was replenished by of the same volume of identical medium. To evaluate the quantities of CUR in the collected samples, UV-Vis absorbance at 420 nm was applied. At the end, cumulative CUR release were plotted during 150 h. 2.6. Cellular toxicity: MTT MTT experiment was performed to assess the cellular toxicity of free CUR and HA-CUR NPs. In brief, CD44 positive (C26) and CD44 negative (CHO) cells were seeded into 96-well plates (5×10 3 cells/well) in RPMI medium supplemented with FBS (10% v/v) and incubated at 37°C under 5% CO 2 atmosphere for 18 h. Then, the cells were either treated with HA-linker (different HA concentrations, 1.17–150 µg/mL), free CUR, and HA-CUR NPs (different CUR concentrations, 1.17–150 µg/mL) for 24h. At the next stage, the media were replaced with fresh medium. Thereafter, MTT solution was transferred to each well and incubated for 3 h. To end, the media were replaced with DMSO. The UV absorbance was recorded at 570 nm (620 nm as a reference) using a microplate reader (Tecan Group Ltd., Switzerland). Moreover, to verify CD44 selective targeting toward C26 cells by HA-CUR NPs, a competition assay was conducted. In this regard, 100 µL of free HA solution (10 mg/mL, hydrated overnight in serum- and antibiotic-free medium) was added to the wells 1h before treating with HA-CUR NPs. 2.7. Cellular internalization 2.7.1. Flow cytometry analysis For cellular uptake investigation of HA-CUR NPs, C26 and CHO cells were seeded into 12-well plates (1×10 5 cells/well). After 18 h incubation, cells were exposed to HA-CUR or free CUR (CUR equivalent concentration 18.75 µg/mL) and incubated for 4 h. After treatment, the cells were trypsinized and washed three times with cold PBS pH 7.4. Finally, the rinsed cells were resuspended in cold PBS (250 µL) and the internalization of the free curcumin or HA-CUR was studied by measuring curcumin fluorescence using BD FACS Calibur TM Flow Cytometer equipped with 488 lasers in the FL1 channel detector. The obtained data were evaluated using FlowJo 7.6 software. To approve CD44 specific targeting toward C26 cells by HA-CUR NPs, a competition assay was performed. In this regard, 800 µL of free HA solution (10 mg/mL, hydrated overnight in serum- and antibiotic-free medium) was added to the wells 1h before treating with HA-CUR NPs. 2.7.2. Fluorescence imaging C26 and CHO cells were seeded in 12-well plates (1×10 5 cells/well) and cultured overnight. Next day, cells were exposed to either HA-CUR or free CUR (18.75 µg/mL CUR) for 4 h, then media were aspirated and cells were rinsed with PBS. At the end, images of cells exposed to either free curcumin or HA-CUR were obtained using inverted fluorescence microscopy. The competition assay was performed as defined in section 2.6.1. 2.8. In vivo therapeutic efficacy The in vivo procedures were conducted in accordance with the EU Directive 2010/63/EU for animal experiments (Approved ethic code in Mashhad University of Medical Sciences: IR.MUMS.PHARMACY.REC.1399.088). To analyze the in vivo antitumor activities of HA-CUR NPs, C26 cells (4×10 5 cells in 80 µL PBS per mouse) was subcutaneously inoculated into the right flank of Female BALB/c mice. The sex of animals did not have any influence on the results of the experiment. After the tumor obtained dimensions of 20 mm 3 in size, the mice were randomly distributed into three groups (n = 5), and inoculated via tail vein with 200 µL of free CUR or HA-CUR (equivalent concentration of 3 mg/kg CUR, all prepared in PBS), every two days for a total of three doses. PBS was injected as a negative control group. It is important to mention that the final concentration of ethanol for preparing free CUR solution was under 5% in all in vitro and in vivo experiments which has no toxicity and it is also safe in human for intravenous injection. Bodyweight, tumor volume, and survival rate were examined for 30 days post-injection until one of the below states for euthanasia was happened: (1) decrease of mice weight less than 20% of their initial weight; (2) mice being ill or unable to feed; (3) tumor volume was bigger than 2 cm 3 , or (4) if they died unexpectedly. The formula derived to calculate tumor volume was (height × length × width × 0.5). 2.9. Ex vivo biodistribution study In order to estimate the biodistribution behavior of the platform, C26 cells (4 × 10 5 cells per mouse in 80 µL PBS), were injected subcutaneously into the right flank of BALB/c mice. Next, mice with tumor size of 200 mm 3 were unsystematically distributed into three groups (PBS control, free CUR, and HA-CUR) and were administered by a single intravenous injection (200 µL 3 mg CUR equiv/kg). Next, 6 and 24 h post injections, mice were sacrificed and heart, spleen, lungs, liver, kidneys, and tumor were removed and imaged using KODAK IS in vivo multispectral imaging apparatus. The biodistribution behavior of free CUR and HA-CUR NPs in C26-tumor bearing mice (Excitation: 470nm, Emission:550 nm) was assessed using KODAK Molecular Imaging® software version 5.0. 2.10. Statistics The statistical significance was analyzed by one-way analysis of variance (ANOVA). It should be noted that P < 0.05 was reflected significant. 3. Results and discussion 3.1. Characterization of the synthesized HA-CUR conjugate HA-CUR conjugate was effectively synthesized in three steps. First, hydroxyl group of EGVE was covalently bonded to 50% of carboxylic acid groups of HA via steric bond formation for obtaining HA-EGVE. Next, the HA-EGVE was coupled to CUR through an acid-cleavable acetal linkage (Fig. 1 A). Successful synthesis of HA-CUR conjugate was confirmed via 1 H NMR and FT-IR spectroscopy. As shown in the 1 H NMR spectrum of HA-CUR conjugate ( Fig. 1 B), the characteristic signals corresponding to HA were detected at 1.8 and 4.8 ppm. The appearance of resonance at 4.56 ppm corresponding to acetal linkage protons verified conjugation of CUR to HA-EGVE. Furthermore, the resonance peaks corresponded to CUR were detected at 6.1–7.9 ppm and 3.86 ppm. The synthesized conjugate was also characterized implementing FT-IR spectroscopy (Fig. 1 C). HA conjugation to CUR by acetal linkage was further confirmed by observation of acetal stretch band at 1077 cm − 1 . Whilst, in the spectrum of HA-CUR conjugate, the elimination of specific band corresponding to the C = O of the carboxylic acid in HA at 1736 cm − 1 , and appearance of sharp strong peaks at 1631 cm − 1 and 1507 cm − 1 which were accredited to the C = O and C = C of CUR further verified the successful synthesis of HA-CUR conjugate. The grafting yield of curcumin to HA-EGVE conjugate was calculated to be 100%, implementing integral of c proton of the CUR and matched it with integral of HA protons. Additionally, it was obtained that 1 mg of HA-CUR contained 668.175 ± 20.57 µg CUR. 3.3. Physicochemical characterization of nanosystem The self-assembled HA-CUR NPs were prepared via direct hydration method. In this system, HA as a hydrophilic biopolymeric back bone and CUR as a lipophilic conjugate provided an appropriate amphiphilic balance to form a self-assembled micellar structure. Dynamic light scattering analysis of the nanoparticles in terms of hydrodynamic diameter, PDI and zeta potential was obtained as 84.21 ± 4.75 nm, 0.321 ± 0.12 and − 22 ± 7.66 mV, respectively. The hydrodynamic diameter of HA-CUR was less than 100 nm. Bearing in mind that EPR (enhanced permeation and retention) effect could be the reason of nanoparticulate accumulation platforms with size of below 200 nm [ 30 ], thus it could improve the pharmacokinetics and therapeutic efficiency of the conjugated CUR. Furthermore, moderate anionic zeta potential is critical for nanoparticles stability and systemic administration safety, in terms of hemolytic and cytotoxic activities [ 31 ]. Due to the presence of HA in the structure of HA-CUR NPs and its abundant carboxylic acid groups, the net charge of the NPs was negative. SEM was applied to assess the morphological characteristics of HA-CUR NPs. Figure 2 shows spherical shape of nanoparticles with 100 nm diameter with appropriate homogeneity. 3.5. CUR release profile The CUR release pattern in two pH values (pH 5.4 and 7.4) was investigated. The release profile showed a significantly accelerated CUR release at pH 5.4 compared to pH 7.4 (Fig. 3 ). After 24 h, ≈ 50% of the CUR was released from the HA-CUR NPs at pH 5.4 versus 18% at pH 7.4. The observed accelerated CUR release at pH 5.4 could be ascribed to the disassembly of acid-cleavable acetal linkage between CUR and the polymer backbone in acidic environment. In this regard, numerous studies reported acetal linkage as a stimulus responsive bond in drug delivery systems including drug conjugates [ 32 , 33 ]. 3.6. Cellular toxicity The survival percentage of C26 and CHO cells which treated with free CUR and HA-CUR NPs was evaluated using MTT assay. The results showed that the C26 cells exposed to various concentrations of either HA-CUR NPs and free CUR exhibited dose-dependent cytotoxicity. However, free CUR showed more cytotoxicity at high concentrations compared to HA-CUR NPs which is mainly due to the higher uptake of free CUR as a small molecule via diffusion mechanism in comparison with endocytosis internalization of HA-CUR NPs [ 34 – 36 ] (Fig. 4 A ) . Moreover, no obvious cytotoxicity for HA-CUR NPs was observed in CHO cells (Fig. 4 B ) . For explaining this phenomenon, it can be said that the HA shell can enhance the transportation of CUR to C26 cells in a selective manner through CD44-mediated endocytosis. As illustrated in Fig. 4 C, cellular toxicity of HA-CUR nanosystems against C26 cells, pretreated with HA was considerably reduced to concentration of 18.75 µg/mL. Thus we chose this concentration for further uptake studies. 3.7. Cellular internalization investigation 3.7.1. Flow cytometry study Selective targeting against cancerous cells is one of the most vital features of a smart drug delivery platform. For evaluation of cellular uptake and targeting capability of HA-CUR NPs toward CD44 positive C26 cells, we compared the internalization of HA-CUR NPs and free CUR after 3 h of incubation using flow cytometry experiment. As shown in Fig. 5 A, B, in CD44 overexpressing C26 cell line, cellular internalization of HA-CUR nanosystems was higher in comparison with CHO cell line, confirming the cell selectivity of the prepared platform. Also, a noteworthy reduction in cellular internalization of HA-CUR NPs by C26 cells pre-exposed to free HA as competing ligand was identified compared to the cells without HA pre-exposure. This finding verified active targeting of CD44 receptor in C26 cells via HA as a ligand. Other studies have also reported the uptake of HA-decorated NPs by CD44-mediated endocytosis [ 24 , 37 , 38 ]. For instance, Shahriari et al. indicated that the HA-coated platform is able to enhance the cellular internalization and cytotoxicity against CD44 overexpressing 4T1 cells [ 26 ]. 3.7.2. Fluorescence imaging For further assessment of cellular internalization of HA-CUR NPs, cellular uptake study on C26 and CHO was carried out using fluorescent microscopy ( Fig. 6 ). We evaluated the cellular uptake between HA-CUR nanosystems and free CUR by both C26 and CHO cells, employing green fluorescence of curcumin. The obtained data illustrated that the fluorescence intensity of C26 cells which were treated with HA-CUR NPs for 4h was remarkably greater than that of CHO cells, confirming superior internalization into C26 cells in comparison with CHO cells. Moreover, for HA-CUR NPs, the fluorescence intensity of the C26 cells pretreated with free HA as a free competing ligand for 1h was significantly decreased compared to those without pretreatment. It should be noted that free HA blocked the CD44 receptor and inhibited extensive cellular uptake of the prepared platform via receptor-mediated endocytosis. 3.8. In vivo anticancer effect of HA-CUR The tumor suppressive effect of HA-CUR NPs in comparison with free CUR was evaluated using C26 tumorized mice. Mice received three doses of HA-CUR nanosystems and free CUR. Findings displayed that HA-CUR nanosystems and free CUR therapy resulted in considerably reduced tumor volumes in comparison with control group (Fig. 7 A). Furthermore, tumor growth suppression potency of HA-CUR nanosystems was impressively higher in comparison with free CUR which could be ascribed to both EPR effect and the selective tumor targeting capability of HA-CUR nanosystems due to the presence of HA shell ( Fig. 7 A ) . Various studies have demonstrated the role of HA as a targeting moiety for guided delivery of anticancer nanomedicines [ 24 , 38 , 39 ]. In this regard, Lai et al. , indicated that the pH-responsive acid-labile hydrazine linkage of HA-CUR conjugate can noticeably improve the antitumor efficacy of murine metastatic breast cancer in vitro and in vivo [ 10 ]. In another study, Seok and co-workers developed curcumin-loaded zein nanoparticles coated with HA. They found that CUR was successfully delivered to CD44-overexpressed CT26 tumor cells and improved the CUR biodistribution in vivo [ 40 ]. The biosafety of the prepared nanoformulation was further investigated by monitoring mice's body weight in each group. The body weight analysis illustrated no obvious changes between all groups, 30 days after intravenous administration ( Fig. 7 B ) . Moreover, the survival percentage investigation demonstrated that five out of five mice treated with HA-CUR NPs survived during 30 days while in free CUR group, four of five mice stayed alive and three of five mice in the control group remained alive up to 30 days (Fig. 7 C). 3.9. Ex vivo fluorescence imaging Organ biodistribution and tumor accumulation of free CUR and HA-CUR nanosustems in colon cancer C26 BALB/c murine tumor model was evaluated ex vivo by imaging of excised tissues, 6 and 24 h post-administration. As depicted in Fig. 8 A tumors displayed highest fluorescence intensities compared to other organs in HA-CUR groups. Fluorescence intensity of each organ was further quantified and the obtained data are represented in Fig. 8 B, C. HA-CUR NPs showed greater tumor uptake and accumulation in comparison with free CUR 6 and 24 h post-injection, demonstrating that HA conjugation enhanced tumor targeting capacity of HA-CUR NPs. Drug conjugation to HA as a therapeutic polymer could improve biocompatibility, prolong the blood circulation profile, and most importantly, impart active targeting capability, associated with CD44 overexpressed on many cancer cells. Thus the drug-polymer conjugate can reach tumors at high concentrations. Moreover, the biocompatibility of the HA as a biodegradable biopolymer allows for multiple injections of the systems for better therapeutic outcomes [ 41 – 43 ]. 4. Conclusion In summary, we developed a pH-sensitive self-assembled CD44-targeted therapeutic micelles comprised of curcumin (CUR)-hyaluronic acid (HA) conjugate. In this system, CUR was conjugated to HA through an acetal linkage. The prepared HA-CUR conjugate self-assembled and formed a micellar structure with size of 84 nm. The prepared platform showed great potency to selectively deliver CUR to CD44-positive, C26 cancer cells in comparison with CD44-negative CHO cell line, confirming the hypothesis that HA amended the guided transportation of NPs. Moreover, in vivo study showed a remarkable therapeutic potency for HA-CUR NPs compared to free CUR in terms of tumor growth suppression, survival rate and biodistribution. It could be concluded that the HA-CUR NPs could be introduced as a promising targeted self-assembled bioconjugate for intelligent delivery of CUR to colon adenocarcinoma. Declarations Acknowledgments We would acknowledge the support of the Mashhad University of Medical Sciences, Mashhad, Iran. (Grant number: 991228). Author Contribution N.H. Methodology, Data analysis, Data curation, Writing-original draft. S.D. Methodology, Data analysis, Data curation, Writing-original draft, Writing-original draft. S.M.T. Visualization, Data Curation. K.A. Visualization, Data Curation. M.R. Writing - review & editing, Funding acquisition, Validation. M.A. Writing - review & editing, Conceptualization, Supervision, Investigation, Validation. All authors read and approved the final manuscript. Funding MR received funding from Mashhad University of Medical Sciences, Mashhad, Iran. (Grant number: 991228). Declarations The in vivo procedures were conducted in accordance with the EU Directive 2010/63/EU for animal experiments (Approved ethic code in Mashhad University of Medical Sciences: IR.MUMS.PHARMACY.REC.1399.088). Conflict of interest The authors declare that they have no confidence of interest. References Yin Z, Yao C, Zhang L, Qi S (2023) Application of artificial intelligence in diagnosis and treatment of colorectal cancer: A novel Prospect. Front Med 10: 1128084. https://doi.org/10.3389/fmed.2023.1128084 Siegel RL, Wagle NS, Cercek A, Smith RA, Jemal A (2023) Colorectal cancer statistics, 2023. 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J Control Release 335: 369-388. https://doi.org/10.1016/j.jconrel.2021.05.039 Shahriari M, Taghdisi SM, Abnous K, Ramezani M, Alibolandi M (2019) Synthesis of hyaluronic acid-based polymersomes for doxorubicin delivery to metastatic breast cancer. Inte J Pharm 572: 118835. https://doi.org/10.1016/j.ijpharm.2019.118835 Zhang M, Xu C, Wen L, Han MK, Xiao B, Zhou J, Zhang Y, Zhang Z, Viennois E, Merlin D (2016) A Hyaluronidase-Responsive Nanoparticle-Based Drug Delivery System for Targeting Colon Cancer Cells. Cancer Res 76: 7208-7218. https://doi.org/10.1158/0008-5472.CAN-16-1681 Donelan W, Dominguez-Gutierrez PR, Kusmartsev S (2022) Deregulated hyaluronan metabolism in the tumor microenvironment drives cancer inflammation and tumor-associated immune suppression. Front Immunol 13: 971278. https://doi.org/10.3389/fimmu.2022.971278 Nelson KM, Dahlin JL, Bisson J, Graham J, Pauli GF, Walters MA (2017) The essential medicinal chemistry of curcumin: miniperspective. J Med Chem 60: 1620-1637. https://doi.org/10.1021/acs.jmedchem.6b00975 Nakamura Y, Mochida A, Choyke PL, Kobayashi H (2016) Nanodrug Delivery: Is the Enhanced Permeability and Retention Effect Sufficient for Curing Cancer?. Bioconjug Chem, 27: 2225-2238. https://doi.org/10.1021/acs.bioconjchem.6b00437 Niroumand U, Firouzabadi N, Goshtasbi G, Hassani B, Ghasemiyeh P, Mohammadi-Samani S. The effect of size, morphology and surface properties of mesoporous silica nanoparticles on pharmacokinetic aspects and potential toxicity concerns. Frontiers in Materials 10: 1189463. https://doi.org/10.3389/fmats.2023.1189463 Gillies ER, Goodwin AP, Fréchet JMJ (2004) Acetals as pH-Sensitive Linkages for Drug Delivery. Bioconjug Chem 15: 1254-1263. Gannimani R, Walvekar P, Naidu VR, Aminabhavi TM, Govender T. Acetal containing polymers as pH-responsive nano-drug delivery systems. J Control Release 328: 736-761. https://doi.org/10.1016/j.jconrel.2020.09.044 Alibolandi M, Taghdisi SM, Ramezani P, Shamili FH, Farzad SA, Abnous K, Ramezani M (2017) Smart AS1411-aptamer conjugated pegylated PAMAM dendrimer for the superior delivery of camptothecin to colon adenocarcinoma in vitro and in vivo. Int J Pharm 519: 352-364. https://doi.org/10.1016/j.ijpharm.2017.01.044 Sahranavard M, Shahriari M, Abnous K, Hadizadeh F, Taghdisi SM, Zolfaghari R, Ramezani M, Alibolandi M (2021) Design and synthesis of targeted star-shaped micelle for guided delivery of camptothecin: In vitro and in vivo evaluation. Mater Sci Eng C 131: 112529. https://doi.org/10.1016/j.msec.2021.112529 Alibolandi M, Hoseini F, Mohammadi M, Ramezani P, Einafshar E, Taghdisi SM, Ramezani M, Abnous K (2018) Curcumin-entrapped MUC-1 aptamer targeted dendrimer-gold hybrid nanostructure as a theranostic system for colon adenocarcinoma. Int J Pharm 549: 67-75. https://doi.org/10.1016/j.ijpharm.2018.07.052 Liu L, Yang S, Chen F, Cheng K-W (2022) Hyaluronic Acid–Zein Core-Shell Nanoparticles Improve the Anticancer Effect of Curcumin Alone or in Combination with Oxaliplatin against Colorectal Cancer via CD44-Mediated Cellular Uptake. Molecules 27: 1498. https://doi.org/10.3390/molecules27051498 Ji P, Wang L, Chen Y, Wang S, Wu Z, Qi X (2020) Hyaluronic acid hydrophilic surface rehabilitating curcumin nanocrystals for targeted breast cancer treatment with prolonged biodistribution. Biomater sci 8: 462-472. https://doi.org/10.1039/C9BM01605H Kaewruethai T, Lin Y, Wang Q, Luckanagul JA (2023) The Dual Modification of PNIPAM and β-Cyclodextrin Grafted on Hyaluronic Acid as Self-Assembled Nanogel for Curcumin Delivery. Polymers 15: 116. https://doi.org/10.3390/polym15010116 Seok H-Y, Sanoj Rejinold N, Lekshmi KM, Cherukula K, Park I-K, Kim Y-C. CD44 targeting biocompatible and biodegradable hyaluronic acid cross-linked zein nanogels for curcumin delivery to cancer cells: In vitro and in vivo evaluation. J Control Release 280: 20-30. https://doi.org/10.1016/j.jconrel.2018.04.050 Chen B, Miller RJ, Dhal PK (2014) Hyaluronic acid-based drug conjugates: state-of-the-art and perspectives. J Biomed Nanotechnol 10: 4-16. https://doi.org/10.1166/jbn.2014.1781 Huang G, Huang H (2018) Application of hyaluronic acid as carriers in drug delivery. Drug Deliv 25: 766-772. https://doi.org/10.1080/10717544.2018.1450910 Fu C-P, Cai X-Y, Chen S-L, Yu H-W, Fang Y, Feng X-C, Zhang L-M, Li C-Y (2023) Hyaluronic Acid-Based Nanocarriers for Anticancer Drug Delivery. Polymers 15: 2317. https://doi.org/10.3390/polym15102317 Additional Declarations No competing interests reported. Supplementary Files SupportinginformationBionanomedicine.docx Cite Share Download PDF Status: Published Journal Publication published 28 Nov, 2024 Read the published version in BioNanoScience → Version 1 posted Editorial decision: Revision requested 26 Jul, 2024 Reviews received at journal 17 Jun, 2024 Reviews received at journal 07 Jun, 2024 Reviewers agreed at journal 28 May, 2024 Reviewers agreed at journal 27 May, 2024 Reviewers invited by journal 27 May, 2024 Editor assigned by journal 06 Apr, 2024 Submission checks completed at journal 05 Apr, 2024 First submitted to journal 04 Apr, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4216826","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":288198699,"identity":"75983412-e884-4792-a6ea-7b241c7f1044","order_by":0,"name":"Niloufar Hazrati","email":"","orcid":"","institution":"Mashhad University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Niloufar","middleName":"","lastName":"Hazrati","suffix":""},{"id":288198700,"identity":"bdd7c132-d58e-4654-9105-6974f1d40be1","order_by":1,"name":"Sadegh Dehghani","email":"","orcid":"","institution":"Mashhad University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Sadegh","middleName":"","lastName":"Dehghani","suffix":""},{"id":288198701,"identity":"0f4f2bec-c919-4840-a4ee-0cb6e7667ff8","order_by":2,"name":"Sahar Taghavi","email":"","orcid":"","institution":"Mashhad University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Sahar","middleName":"","lastName":"Taghavi","suffix":""},{"id":288198702,"identity":"d261923d-dc70-4715-9df6-d6524ed36978","order_by":3,"name":"Seyed Mohammad Taghdisi","email":"","orcid":"","institution":"Mashhad University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Seyed","middleName":"Mohammad","lastName":"Taghdisi","suffix":""},{"id":288198703,"identity":"0d8b8f0b-e542-4710-8bba-b7731d835cec","order_by":4,"name":"Khalil Abnous","email":"","orcid":"","institution":"Mashhad University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Khalil","middleName":"","lastName":"Abnous","suffix":""},{"id":288198704,"identity":"c0843b47-686d-48eb-9e68-eb8500e35d68","order_by":5,"name":"Mohammad Ramezani","email":"","orcid":"","institution":"Mashhad University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Mohammad","middleName":"","lastName":"Ramezani","suffix":""},{"id":288198705,"identity":"296c2b74-8c6f-450c-ab72-7cbce0f3777e","order_by":6,"name":"Mona Alibolandi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAElEQVRIiWNgGAWjYPACCx4DBuYGBgY2CTkQ98ADwlokgFoYwVqMwVoSiNDCANXCkNgA4uPTotvee/ADQ42EjDl7Y+PngjKL9Plhhx8CbbGT023ArsXszLlkCYZjEjyWPQebpWeck8jdeDvNAKgl2djsAA4tN3IMJIBe4DG4kdggzdsG1DI7AaTlQOI23FqMfzD8A2tp/g3Ukm44O/0DIS1mEoxtYC1tIFsS5KVzCNhy5oyZRWIf2C9t1jznJAw3SOcUHEgwwOOX4z3GNz58s7E3Z28+fJunrE5efnb65g8fKuzkcGkBgwRkjgFYpQEe5RhAvoEU1aNgFIyCUTASAADNsVzuhwdDDwAAAABJRU5ErkJggg==","orcid":"","institution":"Mashhad University of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Mona","middleName":"","lastName":"Alibolandi","suffix":""}],"badges":[],"createdAt":"2024-04-04 09:24:50","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4216826/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4216826/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12668-024-01702-8","type":"published","date":"2024-11-28T15:57:17+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":54367213,"identity":"7749f822-0c75-4393-b29f-945028375eea","added_by":"auto","created_at":"2024-04-09 12:41:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5131321,"visible":true,"origin":"","legend":"\u003cp\u003eSynthesis of HA-CUR. HA was bonded to a linker (EGVE) and then CUR was linked to HA-EGVE, resulting in HA-CUR(A).H NMR spectrum of HA-CUR nanosystem (B), FTIR spectrum of CUR, HA, and HA-CUR (C).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4216826/v1/8e219bd44d76490e9c335678.png"},{"id":54367215,"identity":"de89dc1d-5f61-431a-ab3e-ad96d0fef1e8","added_by":"auto","created_at":"2024-04-09 12:41:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":18160820,"visible":true,"origin":"","legend":"\u003cp\u003eSEM images of HA-CUR nanoplatforms.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4216826/v1/70860c7e5a0ddbeafd845a79.png"},{"id":54367216,"identity":"fe4798ba-f642-4b08-b0e9-211046306f41","added_by":"auto","created_at":"2024-04-09 12:41:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3296572,"visible":true,"origin":"","legend":"\u003cp\u003eRelease profile of CUR from HA-CUR NPs in imitation of physiological (pH 7.4) and tumor microenvironment (pH 5.4) conditions.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4216826/v1/dc2442e46ab9807e937fb002.png"},{"id":54367219,"identity":"9ab26729-1889-4acf-9a73-b28f27393602","added_by":"auto","created_at":"2024-04-09 12:41:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3574803,"visible":true,"origin":"","legend":"\u003cp\u003eViability of C26 (A) and CHO (B) cell lines treated with CUR and HA-CUR. Viability of C26 and CHO cell lines treated with HA-linker (C). Viability of C26 cells exposed to HA-CUR after pretreatment with or without HA (D). All evaluations were based on quadruplicate and error bars were given as SD. Non-significance and significance were set at p\u0026gt;0.5 (ns), *p \u0026lt; 0.05**p \u0026lt; 0.01, *** p \u0026lt; 0.001 and **** p\u0026lt;0.0001 respectively.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4216826/v1/b863132c3cde056a4e75c772.png"},{"id":54367799,"identity":"ed9c0100-e53b-471c-b35e-5792eeff8a47","added_by":"auto","created_at":"2024-04-09 12:49:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4415454,"visible":true,"origin":"","legend":"\u003cp\u003eFlow cytometry evaluation and mean intensity of C26 (A, C) and CHO (B, D) cell lines after 3 h of incubation with either free CUR, HA-CUR, or HA-CUR with HA pretreatment. All tests were performed in triplicates and error bars were given as SD. Non-significance and significance were set at p\u0026gt;0.5 (ns) ,*p \u0026lt; 0.05**p \u0026lt; 0.01, *** p \u0026lt; 0.001 and **** p\u0026lt;0.0001 respectively.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4216826/v1/bea8c5a884bd13478d825e6b.png"},{"id":54367218,"identity":"44a5a829-e088-4894-b5a5-78bc3ec3335e","added_by":"auto","created_at":"2024-04-09 12:41:38","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":9771977,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence imaging of C26 and CHO cell lines treated with HA-CUR nanosystems, free CUR, and HA-CUR nanosystems pretreated with HA.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4216826/v1/14a74a16c8f7c3c48fba1bb9.png"},{"id":54367217,"identity":"ef80c836-145d-47c9-936f-5199015417c3","added_by":"auto","created_at":"2024-04-09 12:41:38","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2750928,"visible":true,"origin":"","legend":"\u003cp\u003eTumor growth inhibitory efficacy (A), body weight of tumor-bearing mice (c), and survival curve (C) of HA-CUR compared with PBS (control), and free CUR groups (n=5, mean± SD).\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4216826/v1/26bd4e11628a624690d216fb.png"},{"id":54367220,"identity":"53adc248-77d0-4224-a36f-63c2c1be0d15","added_by":"auto","created_at":"2024-04-09 12:41:39","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":11495062,"visible":true,"origin":"","legend":"\u003cp\u003eThe fluorescence imaging of tumor and different organs along with fluorescence intensity charts 6 h (A, C) and 24 h (B, D) post-administration of PBS (control), free CUR, and HA-CUR NPs. All tests were performed in triplicates and error bars were given as SD. (*p \u0026lt; 0.05and *** p \u0026lt; 0.001)\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-4216826/v1/463dc10383a87b86b2ea651e.png"},{"id":54368962,"identity":"fd137ee9-a5eb-414e-84fb-34f805be187c","added_by":"auto","created_at":"2024-04-09 12:57:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3445743,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4216826/v1/486ba6f9-65b1-4038-ad6c-8d5e0d19a37c.pdf"},{"id":54367212,"identity":"fdb24705-b547-4baf-9f2e-d7b5168bb11e","added_by":"auto","created_at":"2024-04-09 12:41:37","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":13859,"visible":true,"origin":"","legend":"","description":"","filename":"SupportinginformationBionanomedicine.docx","url":"https://assets-eu.researchsquare.com/files/rs-4216826/v1/df170d889c3d0dd104132cf2.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Synthesis of a smart pH sensitive micelle containing hyaluronic acid-curcumin bioconjugate against colorectal cancer","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eColorectal cancer (CRC) is known as one of the deadliest cancer in both genders, worldwide. In 2023, approximately 153,020 new cases have recognized with CRC and this disease caused the death of 52,520 individuals [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDifferent conventional treatment options have been implemented for CRC such as chemotherapy, radiation therapy, and surgery, but these treatments showed undesirable adverse effects which can significantly affect patients' quality of life [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Additionally, cancer cells can develop resistance to chemotherapy and radiation, causing the failure of chemo and radio-therapy [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs a therapeutic strategy to improve cancer treatment outcomes, scientists have been exploring alternative therapeutics, including natural compounds comprising curcumin, resveratrol, lycopene and gingerol. These compounds have been found to have chemopreventive and/or anticancer properties with minimal side effects [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this regard, curcumin as a natural polyphenol and the active component of a medical plant named \u003cem\u003eCurcuma longa\u003c/em\u003e has shown promising antimicrobial, anti-inflammatory and wound healing activities [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Furthermore, numerous \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experiments reported anti-tumor characteristics for curcumin against lung cancer, pancreatic cancer, brain tumors, cervical cancer, and colorectal cancer [\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, low aqueous solubility, poor bioavailability, rapid metabolism and chemical instability of curcumin have restricted its therapeutic efficacy [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOver the past years, various nanotechnology-based drug delivery systems were designed to improve curcumin pharmacokinetics and overcome the aforementioned limitations toward systemic delivery. These nanoparticulate systems such as nanoemulsion, solid lipid nanoparticles, phytosomes, gold nanoparticles, liposomes, and polymersomes improved therapeutic index of curcumin [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAmong different nanoplatforms, polymer-drug conjugates or therapeutic polymers are promising platforms which could increase solubility and stability of curcumin while improving its pharmacokinetics.\u003c/p\u003e \u003cp\u003eIn these systems, stimuli-sensitive linkage between polymer and drug is formed which responds to inherent or external environmental stimuli such as reactive oxygen species, light, ultrasound, temperature, specific enzymes, and pH, thereby providing an intelligent smart platform [\u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Inserting of acetal linkage as pH-sensitive bond between polymer back bone and drug can control and allow triggered release of chemotherapeutic agent from the conjugate at the intended site, under acidic environment in lysosomes and tumor tissues, enhancing the therapeutic efficiency and diminishing chemotherapy related side effects [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHA is known as a biodegradable natural polymer with a linear structure consisted of alternating repeats of N-acetyl-D-glucosamine and D-glucuronic acid [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. HA can be chemically modified through its carboxylic acid and hydroxyl groups [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. HA-drug conjugates are suitable drug delivery platforms for different therapeutic agents due to the biocompatibility, nontoxicity, nonimmunogenicity and targeting capability of HA to CD44 receptor which is overexpressed in a majority of cancerous cells [\u003cspan additionalcitationids=\"CR24 CR25\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Moreover, HA is degraded by hyaluronidases, such as HYAL1, resulting in low molecular weight components after internalization into cancer cells through CD44 receptor-mediated endocytosis [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe current work reports on the synthesis of a novel multifunctional nanoplatform derived from HA conjugated with curcumin (HA-CUR) through pH-sensitive acetal linkage for on-demand release and active targeting of CD44 receptors against colorectal cancer.\u003c/p\u003e \u003cp\u003eThe current study investigated the hypothesis that this therapeutic pH responsive polymer-drug conjugate could self-assemble to micellar structure and the hyaluronic acid as a targeting ligand on the micellar surface could enhance the cellular uptake and anti-cancer potency of synthesized HA-CUR nanoplatform through CD44-mediated endocytosis. Various experiments were conducted to characterize the structure, size, release kinetics, anticancer activity, and intracellular entrance of the prepared nanoplatform. Moreover, biodistribution and the therapeutic index of HA-CUR nanoplatform were investigated \u003cem\u003ein vivo\u003c/em\u003e on subcutaneous C26 murine colon cancer model.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials\u003c/h2\u003e \u003cp\u003eSee supporting information for details.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. HA-CUR conjugate synthesis\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1. Characterization\u003c/h2\u003e \u003cp\u003eIn each step of the synthesis procedure, the product in deuterated solvents were characterized by \u003csup\u003e1\u003c/sup\u003eHNMR (proton nuclear magnetic resonance) spectroscopy using \u003csup\u003e1\u003c/sup\u003eHNMR spectrometer (Bruker, Rheinstetten, Germany). The Perkin Elmer FTIR spectrometer was used to obtain Infrared spectra (FTIR) of HA, CUR and HA-CUR conjugate in KBr pellets.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2. Sodium removal from sodium hyaluronate\u003c/h2\u003e \u003cp\u003eFor sodium removal from sodium hyaluronate, sodium hyaluronate (500 mg) was dissolved in 10 mL of HCl (0.1 N), followed by stirring for 3h at room temperature. Afterwards, it was placed into a dialysis tube (M\u003csub\u003eW\u003c/sub\u003e cut off =\u0026thinsp;2 kD) overnight against one liter of deionized water at 4 \u003csup\u003e◦\u003c/sup\u003eC. Ultimately, the white powder of purified sodium-free HA was obtained by freeze-drying and stored at \u0026minus;\u0026thinsp;20\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.2.3. Conjugation of HA to EGVE\u003c/h2\u003e \u003cp\u003eIn the next step, sodium-free HA was conjugated to the ethylene glycol vinyl ether (EGVE) linker (equivalent to 50% of carboxylic acid groups of HA). For this purpose, 100 mg of sodium-free HA was dissolved in 10 mL anhydrous DMSO, then DMAP was added (molar ratio of EGVE to DMAP was 1:4) to the mixture and stirred for 10 min. Next, DCC was added (molar ration of DMAP:DCC was 1:1) and further stirred for 2 h to make active carboxylic acids of HA. Then, EGVE was added and gently stirred overnight at 50\u0026deg;C. In the next step, under vacuum the solvent was removed and the suspension of HA-EGVE was poured into dialysis tubing with a cut off of 2 kD in order to dialyzed against distilled water for 24 h. Finally, the HA-EGVE was lyophilized and stored at -20\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.2.4 Conjugation of curcumin to the HA-EGVE\u003c/h2\u003e \u003cp\u003eFor fabrication of pH-sensitive acetal bond between EGVE and CUR, 6 mL of DMSO was added to HA-EGVE (100 mg) and sonicated for 30 min at 60\u0026deg;C. Next, in order to functionalize the synthesized HA-EGVE, \u003cem\u003ep\u003c/em\u003e-toluenesulfonic acid monohydrate and CUR (molar ratio of \u003cem\u003ep\u003c/em\u003e-toluenesulfonic acid monohydrate: EGVE:CUR was 1.5:1:1) were added and stirred overnight. The resultant product was dialyzed using dialysis tube with 2 kDa cut off against distilled water overnight. Then, the HA-CUR was lyophilized and kept at -20\u0026deg;C. All synthesis steps were conducted under dark conditions to avoid CUR degradation [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Self-assembly of HA-CUR\u003c/h2\u003e \u003cp\u003eHA-CUR conjugate was self-assembled through direct hydration technique. For this purpose, one mg of HA-CUR conjugate was dispersed in distilled water and bath sonicated for 15 min. The content of the conjugated curcumin was calculated, implementing the UV absorbance of CUR at 420 nm (Varian CARY 100 spectrophotometer, California, USA). To this aim, 1 mL of DMSO was added to 1 mg of HA-CUR and the UV absorption of the solution was measured at 420 nm. Then, the CUR content was calculated using standard curve of CUR.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Morphological characteristics\u003c/h2\u003e \u003cp\u003eTo investigate the morphological features and size of the HA-CUR NPs in deionized water (100 \u0026micro;g/1mL), with field emission scanning electron microscope (FESEM), using TESCAN BRNO- Mira3 LMU instrument made in Czech Republic and dynamic Light-Scattering (DLS) Instrument-Malvern Instruments Zetasizer NanoSampler (Malvern, United Kingdom).\u003c/p\u003e \u003cp\u003eTo this aim, HA-CUR NPs (1 mg/mL) were poured on an aluminum stubs, dehydrated, and then covered with a thin layer of gold to create the connection with the aluminum surface. Finally, the imaging procedure was operated at 10 kV.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.5. \u003cem\u003eCUR\u003c/em\u003e release profile\u003c/h2\u003e \u003cp\u003eFor investigation of CUR \u003cem\u003ein vitro\u003c/em\u003e release from HA-CUR NPs, dialysis method was implemented. In this regard, 1 mL of HA-CUR suspension was poured into two dialysis sacs with M\u003csub\u003eW\u003c/sub\u003e cut off =\u0026thinsp;2 kD, immersed in either 25 mL of PBS (pH 7.4) or citrate buffer (pH 5.5), supplemented with 0.5% tween 80 and maintained in a shaker incubator (37\u0026deg;C, 80 rpm). The release media were protected from light during the experiment. At predesigned times, 1 mL of each vessel was taken out and was replenished by of the same volume of identical medium. To evaluate the quantities of CUR in the collected samples, UV-Vis absorbance at 420 nm was applied. At the end, cumulative CUR release were plotted during 150 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Cellular toxicity: MTT\u003c/h2\u003e \u003cp\u003eMTT experiment was performed to assess the cellular toxicity of free CUR and HA-CUR NPs. In brief, CD44 positive (C26) and CD44 negative (CHO) cells were seeded into 96-well plates (5\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells/well) in RPMI medium supplemented with FBS (10% v/v) and incubated at 37\u0026deg;C under 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere for 18 h. Then, the cells were either treated with HA-linker (different HA concentrations, 1.17\u0026ndash;150 \u0026micro;g/mL), free CUR, and HA-CUR NPs (different CUR concentrations, 1.17\u0026ndash;150 \u0026micro;g/mL) for 24h. At the next stage, the media were replaced with fresh medium. Thereafter, MTT solution was transferred to each well and incubated for 3 h. To end, the media were replaced with DMSO. The UV absorbance was recorded at 570 nm (620 nm as a reference) using a microplate reader (Tecan Group Ltd., Switzerland). Moreover, to verify CD44 selective targeting toward C26 cells by HA-CUR NPs, a competition assay was conducted. In this regard, 100 \u0026micro;L of free HA solution (10 mg/mL, hydrated overnight in serum- and antibiotic-free medium) was added to the wells 1h before treating with HA-CUR NPs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Cellular internalization\u003c/h2\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.7.1. Flow cytometry analysis\u003c/h2\u003e \u003cp\u003eFor cellular uptake investigation of HA-CUR NPs, C26 and CHO cells were seeded into 12-well plates (1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well). After 18 h incubation, cells were exposed to HA-CUR or free CUR (CUR equivalent concentration 18.75 \u0026micro;g/mL) and incubated for 4 h. After treatment, the cells were trypsinized and washed three times with cold PBS pH 7.4. Finally, the rinsed cells were resuspended in cold PBS (250 \u0026micro;L) and the internalization of the free curcumin or HA-CUR was studied by measuring curcumin fluorescence using BD FACS Calibur TM Flow Cytometer equipped with 488 lasers in the FL1 channel detector. The obtained data were evaluated using FlowJo 7.6 software.\u003c/p\u003e \u003cp\u003eTo approve CD44 specific targeting toward C26 cells by HA-CUR NPs, a competition assay was performed. In this regard, 800 \u0026micro;L of free HA solution (10 mg/mL, hydrated overnight in serum- and antibiotic-free medium) was added to the wells 1h before treating with HA-CUR NPs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.7.2. Fluorescence imaging\u003c/h2\u003e \u003cp\u003eC26 and CHO cells were seeded in 12-well plates (1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well) and cultured overnight. Next day, cells were exposed to either HA-CUR or free CUR (18.75 \u0026micro;g/mL CUR) for 4 h, then media were aspirated and cells were rinsed with PBS. At the end, images of cells exposed to either free curcumin or HA-CUR were obtained using inverted fluorescence microscopy. The competition assay was performed as defined in section 2.6.1.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.8. \u003cem\u003eIn vivo\u003c/em\u003e therapeutic efficacy\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003ein vivo\u003c/em\u003e procedures were conducted in accordance with the EU Directive 2010/63/EU for animal experiments (Approved ethic code in Mashhad University of Medical Sciences: IR.MUMS.PHARMACY.REC.1399.088). To analyze the \u003cem\u003ein vivo\u003c/em\u003e antitumor activities of HA-CUR NPs, C26 cells (4\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells in 80 \u0026micro;L PBS per mouse) was subcutaneously inoculated into the right flank of Female BALB/c mice. The sex of animals did not have any influence on the results of the experiment.\u003c/p\u003e \u003cp\u003eAfter the tumor obtained dimensions of 20 mm\u003csup\u003e3\u003c/sup\u003e in size, the mice were randomly distributed into three groups (n\u0026thinsp;=\u0026thinsp;5), and inoculated \u003cem\u003evia\u003c/em\u003e tail vein with 200 \u0026micro;L of free CUR or HA-CUR (equivalent concentration of 3 mg/kg CUR, all prepared in PBS), every two days for a total of three doses. PBS was injected as a negative control group. It is important to mention that the final concentration of ethanol for preparing free CUR solution was under 5% in all \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experiments which has no toxicity and it is also safe in human for intravenous injection. Bodyweight, tumor volume, and survival rate were examined for 30 days post-injection until one of the below states for euthanasia was happened: (1) decrease of mice weight less than 20% of their initial weight; (2) mice being ill or unable to feed; (3) tumor volume was bigger than 2 cm\u003csup\u003e3\u003c/sup\u003e, or (4) if they died unexpectedly. The formula derived to calculate tumor volume was (height \u0026times; length \u0026times; width \u0026times; 0.5).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.9. \u003cem\u003eEx vivo\u003c/em\u003e biodistribution study\u003c/h2\u003e \u003cp\u003eIn order to estimate the biodistribution behavior of the platform, C26 cells (4 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per mouse in 80 \u0026micro;L PBS), were injected subcutaneously into the right flank of BALB/c mice. Next, mice with tumor size of 200 mm\u003csup\u003e3\u003c/sup\u003e were unsystematically distributed into three groups (PBS control, free CUR, and HA-CUR) and were administered by a single intravenous injection (200 \u0026micro;L 3 mg CUR equiv/kg). Next, 6 and 24 h post injections, mice were sacrificed and heart, spleen, lungs, liver, kidneys, and tumor were removed and imaged using KODAK IS \u003cem\u003ein vivo\u003c/em\u003e multispectral imaging apparatus. The biodistribution behavior of free CUR and HA-CUR NPs in C26-tumor bearing mice (Excitation: 470nm, Emission:550 nm) was assessed using KODAK Molecular Imaging\u0026reg; software version 5.0.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Statistics\u003c/h2\u003e \u003cp\u003eThe statistical significance was analyzed by one-way analysis of variance (ANOVA). It should be noted that P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was reflected significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Characterization of the synthesized HA-CUR conjugate\u003c/h2\u003e \u003cp\u003eHA-CUR conjugate was effectively synthesized in three steps. First, hydroxyl group of EGVE was covalently bonded to 50% of carboxylic acid groups of HA via steric bond formation for obtaining HA-EGVE. Next, the HA-EGVE was coupled to CUR through an acid-cleavable acetal linkage (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Successful synthesis of HA-CUR conjugate was confirmed \u003cem\u003evia\u003c/em\u003e \u003csup\u003e1\u003c/sup\u003eH NMR and FT-IR spectroscopy. As shown in the \u003csup\u003e1\u003c/sup\u003eH NMR spectrum of HA-CUR conjugate \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), the characteristic signals corresponding to HA were detected at 1.8 and 4.8 ppm. The appearance of resonance at 4.56 ppm corresponding to acetal linkage protons verified conjugation of CUR to HA-EGVE. Furthermore, the resonance peaks corresponded to CUR were detected at 6.1\u0026ndash;7.9 ppm and 3.86 ppm.\u003c/p\u003e \u003cp\u003eThe synthesized conjugate was also characterized implementing FT-IR spectroscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). HA conjugation to CUR by acetal linkage was further confirmed by observation of acetal stretch band at 1077 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Whilst, in the spectrum of HA-CUR conjugate, the elimination of specific band corresponding to the C\u0026thinsp;=\u0026thinsp;O of the carboxylic acid in HA at 1736 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and appearance of sharp strong peaks at 1631 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1507 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e which were accredited to the C\u0026thinsp;=\u0026thinsp;O and C\u0026thinsp;=\u0026thinsp;C of CUR further verified the successful synthesis of HA-CUR conjugate. The grafting yield of curcumin to HA-EGVE conjugate was calculated to be 100%, implementing integral of c proton of the CUR and matched it with integral of HA protons. Additionally, it was obtained that 1 mg of HA-CUR contained 668.175\u0026thinsp;\u0026plusmn;\u0026thinsp;20.57 \u0026micro;g CUR.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Physicochemical characterization of nanosystem\u003c/h2\u003e \u003cp\u003eThe self-assembled HA-CUR NPs were prepared \u003cem\u003evia\u003c/em\u003e direct hydration method. In this system, HA as a hydrophilic biopolymeric back bone and CUR as a lipophilic conjugate provided an appropriate amphiphilic balance to form a self-assembled micellar structure. Dynamic light scattering analysis of the nanoparticles in terms of hydrodynamic diameter, PDI and zeta potential was obtained as 84.21\u0026thinsp;\u0026plusmn;\u0026thinsp;4.75 nm, 0.321\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 and \u0026minus;\u0026thinsp;22\u0026thinsp;\u0026plusmn;\u0026thinsp;7.66 mV, respectively.\u003c/p\u003e \u003cp\u003eThe hydrodynamic diameter of HA-CUR was less than 100 nm. Bearing in mind that EPR (enhanced permeation and retention) effect could be the reason of nanoparticulate accumulation platforms with size of below 200 nm [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], thus it could improve the pharmacokinetics and therapeutic efficiency of the conjugated CUR. Furthermore, moderate anionic zeta potential is critical for nanoparticles stability and systemic administration safety, in terms of hemolytic and cytotoxic activities [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDue to the presence of HA in the structure of HA-CUR NPs and its abundant carboxylic acid groups, the net charge of the NPs was negative. SEM was applied to assess the morphological characteristics of HA-CUR NPs. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows spherical shape of nanoparticles with 100 nm diameter with appropriate homogeneity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.5. CUR release profile\u003c/h2\u003e \u003cp\u003eThe CUR release pattern in two pH values (pH 5.4 and 7.4) was investigated. The release profile showed a significantly accelerated CUR release at pH 5.4 compared to pH 7.4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e After 24 h, \u0026asymp;\u0026thinsp;50% of the CUR was released from the HA-CUR NPs at pH 5.4 versus 18% at pH 7.4. The observed accelerated CUR release at pH 5.4 could be ascribed to the disassembly of acid-cleavable acetal linkage between CUR and the polymer backbone in acidic environment. In this regard, numerous studies reported acetal linkage as a stimulus responsive bond in drug delivery systems including drug conjugates [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Cellular toxicity\u003c/h2\u003e \u003cp\u003eThe survival percentage of C26 and CHO cells which treated with free CUR and HA-CUR NPs was evaluated using MTT assay. The results showed that the C26 cells exposed to various concentrations of either HA-CUR NPs and free CUR exhibited dose-dependent cytotoxicity. However, free CUR showed more cytotoxicity at high concentrations compared to HA-CUR NPs which is mainly due to the higher uptake of free CUR as a small molecule \u003cem\u003evia\u003c/em\u003e diffusion mechanism in comparison with endocytosis internalization of HA-CUR NPs [\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Moreover, no obvious cytotoxicity for HA-CUR NPs was observed in CHO cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. For explaining this phenomenon, it can be said that the HA shell can enhance the transportation of CUR to C26 cells in a selective manner through CD44-mediated endocytosis. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, cellular toxicity of HA-CUR nanosystems against C26 cells, pretreated with HA was considerably reduced to concentration of 18.75 \u0026micro;g/mL. Thus we chose this concentration for further uptake studies.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Cellular internalization investigation\u003c/h2\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003e3.7.1. Flow cytometry study\u003c/h2\u003e \u003cp\u003eSelective targeting against cancerous cells is one of the most vital features of a smart drug delivery platform. For evaluation of cellular uptake and targeting capability of HA-CUR NPs toward CD44 positive C26 cells, we compared the internalization of HA-CUR NPs and free CUR after 3 h of incubation using flow cytometry experiment. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B, in CD44 overexpressing C26 cell line, cellular internalization of HA-CUR nanosystems was higher in comparison with CHO cell line, confirming the cell selectivity of the prepared platform. Also, a noteworthy reduction in cellular internalization of HA-CUR NPs by C26 cells pre-exposed to free HA as competing ligand was identified compared to the cells without HA pre-exposure. This finding verified active targeting of CD44 receptor in C26 cells \u003cem\u003evia\u003c/em\u003e HA as a ligand. Other studies have also reported the uptake of HA-decorated NPs by CD44-mediated endocytosis [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. For instance, Shahriari \u003cem\u003eet al.\u003c/em\u003e indicated that the HA-coated platform is able to enhance the cellular internalization and cytotoxicity against CD44 overexpressing 4T1 cells [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003e3.7.2. Fluorescence imaging\u003c/h2\u003e \u003cp\u003eFor further assessment of cellular internalization of HA-CUR NPs, cellular uptake study on C26 and CHO was carried out using fluorescent microscopy \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e We evaluated the cellular uptake between HA-CUR nanosystems and free CUR by both C26 and CHO cells, employing green fluorescence of curcumin. The obtained data illustrated that the fluorescence intensity of C26 cells which were treated with HA-CUR NPs for 4h was remarkably greater than that of CHO cells, confirming superior internalization into C26 cells in comparison with CHO cells. Moreover, for HA-CUR NPs, the fluorescence intensity of the C26 cells pretreated with free HA as a free competing ligand for 1h was significantly decreased compared to those without pretreatment. It should be noted that free HA blocked the CD44 receptor and inhibited extensive cellular uptake of the prepared platform via receptor-mediated endocytosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.8. \u003cem\u003eIn vivo anticancer effect of\u003c/em\u003e HA-CUR\u003c/h2\u003e \u003cp\u003eThe tumor suppressive effect of HA-CUR NPs in comparison with free CUR was evaluated using C26 tumorized mice. Mice received three doses of HA-CUR nanosystems and free CUR. Findings displayed that HA-CUR nanosystems and free CUR therapy resulted in considerably reduced tumor volumes in comparison with control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Furthermore, tumor growth suppression potency of HA-CUR nanosystems was impressively higher in comparison with free CUR which could be ascribed to both EPR effect and the selective tumor targeting capability of HA-CUR nanosystems due to the presence of HA shell \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Various studies have demonstrated the role of HA as a targeting moiety for guided delivery of anticancer nanomedicines [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In this regard, Lai \u003cem\u003eet al.\u003c/em\u003e, indicated that the pH-responsive acid-labile hydrazine linkage of HA-CUR conjugate can noticeably improve the antitumor efficacy of murine metastatic breast cancer \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In another study, Seok and co-workers developed curcumin-loaded zein nanoparticles coated with HA. They found that CUR was successfully delivered to CD44-overexpressed CT26 tumor cells and improved the CUR biodistribution \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe biosafety of the prepared nanoformulation was further investigated by monitoring mice's body weight in each group. The body weight analysis illustrated no obvious changes between all groups, 30 days after intravenous administration \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Moreover, the survival percentage investigation demonstrated that five out of five mice treated with HA-CUR NPs survived during 30 days while in free CUR group, four of five mice stayed alive and three of five mice in the control group remained alive up to 30 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e3.9. \u003cem\u003eEx vivo\u003c/em\u003e fluorescence imaging\u003c/h2\u003e \u003cp\u003eOrgan biodistribution and tumor accumulation of free CUR and HA-CUR nanosustems in colon cancer C26 BALB/c murine tumor model was evaluated \u003cem\u003eex vivo\u003c/em\u003e by imaging of excised tissues, 6 and 24 h post-administration. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA tumors displayed highest fluorescence intensities compared to other organs in HA-CUR groups. Fluorescence intensity of each organ was further quantified and the obtained data are represented in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB, C. HA-CUR NPs showed greater tumor uptake and accumulation in comparison with free CUR 6 and 24 h post-injection, demonstrating that HA conjugation enhanced tumor targeting capacity of HA-CUR NPs. Drug conjugation to HA as a therapeutic polymer could improve biocompatibility, prolong the blood circulation profile, and most importantly, impart active targeting capability, associated with CD44 overexpressed on many cancer cells. Thus the drug-polymer conjugate can reach tumors at high concentrations. Moreover, the biocompatibility of the HA as a biodegradable biopolymer allows for multiple injections of the systems for better therapeutic outcomes [\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn summary, we developed a pH-sensitive self-assembled CD44-targeted therapeutic micelles comprised of curcumin (CUR)-hyaluronic acid (HA) conjugate. In this system, CUR was conjugated to HA through an acetal linkage. The prepared HA-CUR conjugate self-assembled and formed a micellar structure with size of 84 nm.\u003c/p\u003e \u003cp\u003eThe prepared platform showed great potency to selectively deliver CUR to CD44-positive, C26 cancer cells in comparison with CD44-negative CHO cell line, confirming the hypothesis that HA amended the guided transportation of NPs. Moreover, \u003cem\u003ein vivo\u003c/em\u003e study showed a remarkable therapeutic potency for HA-CUR NPs compared to free CUR in terms of tumor growth suppression, survival rate and biodistribution. It could be concluded that the HA-CUR NPs could be introduced as a promising targeted self-assembled bioconjugate for intelligent delivery of CUR to colon adenocarcinoma.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would acknowledge the support of the Mashhad University of Medical Sciences, Mashhad, Iran. \u0026nbsp;(Grant number: 991228).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eN.H. Methodology, Data analysis, Data curation, Writing-original draft. S.D. Methodology, Data analysis, Data curation, Writing-original draft, Writing-original draft. S.M.T. Visualization, Data Curation. K.A. Visualization, Data Curation. M.R. Writing - review \u0026amp; editing, Funding acquisition, Validation. M.A. Writing - review \u0026amp; editing, Conceptualization, Supervision, Investigation, Validation. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMR received funding from Mashhad University of Medical Sciences, Mashhad, Iran. \u0026nbsp;(Grant number: 991228).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe in vivo procedures were conducted in accordance with the EU Directive 2010/63/EU for animal experiments (Approved ethic code in Mashhad University of Medical Sciences: IR.MUMS.PHARMACY.REC.1399.088).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no confidence of interest.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eYin Z, Yao C, Zhang L, Qi S (2023) Application of artificial intelligence in diagnosis and treatment of colorectal cancer: A novel Prospect. 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Polymers 15: 2317. https://doi.org/10.3390/polym15102317 \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bionanoscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bnsc","sideBox":"Learn more about [BioNanoScience](http://link.springer.com/journal/12668)","snPcode":"12668","submissionUrl":"https://submission.nature.com/new-submission/12668/3","title":"BioNanoScience","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Curcumin, Colorectal cancer, Hyaluronic acid, Targeted drug delivery","lastPublishedDoi":"10.21203/rs.3.rs-4216826/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4216826/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn the current study, we fabricated a pH-sensitive self-assembled CD44-targeted therapeutic micelle, comprising curcumin (CUR)-hyaluronic acid (HA) conjugate. At the first stage, the biopolymer, HA, as a back bone was attached to ethylene glycol vinyl ether (equivalent to 50% of the carboxylic acids of HA) and then hydroxyl of curcumin was attached to this linker to form a pH-responsive acetal linkage. The prepared HA-CUR conjugate was self-assembled and formed a micellar structure with size of 84 nm. The release of CUR from the prepared platform illustrated a controlled, sustained release at pH 7.4 while it was significantly accelerated at pH 5.4. The cytotoxicity and cellular uptake of the platform were evaluated against C26 as a CD44 positive and CHO as CD44 negative cells. The cytotoxicity and cellular uptake study showed higher internalization and cellular toxicity of the synthesized platform in C26 cells compared with CHO cells. \u003cem\u003eIn vivo\u003c/em\u003e study demonstrated desirable therapeutic efficacy of HA-CUR toward C26 tumor growth suppression and survival rate of BALB/c mice. These findings suggested HA-CUR as a hopeful natural product-based nanomedicine for active targeting and delivery of CUR to colon adenocarcinoma.\u003c/p\u003e","manuscriptTitle":"Synthesis of a smart pH sensitive micelle containing hyaluronic acid-curcumin bioconjugate against colorectal cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-09 12:41:21","doi":"10.21203/rs.3.rs-4216826/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-26T07:52:36+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-17T10:17:21+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-08T03:17:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"199103582254756606550173050869424397671","date":"2024-05-28T07:45:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"275290522780593218691444918489398635555","date":"2024-05-27T15:26:07+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-27T14:17:06+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-06T10:25:20+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-04-05T05:52:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"BioNanoScience","date":"2024-04-04T09:23:34+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bionanoscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bnsc","sideBox":"Learn more about [BioNanoScience](http://link.springer.com/journal/12668)","snPcode":"12668","submissionUrl":"https://submission.nature.com/new-submission/12668/3","title":"BioNanoScience","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"945bf6cb-972f-4d37-828e-34634d563217","owner":[],"postedDate":"April 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-02T16:00:52+00:00","versionOfRecord":{"articleIdentity":"rs-4216826","link":"https://doi.org/10.1007/s12668-024-01702-8","journal":{"identity":"bionanoscience","isVorOnly":false,"title":"BioNanoScience"},"publishedOn":"2024-11-28 15:57:17","publishedOnDateReadable":"November 28th, 2024"},"versionCreatedAt":"2024-04-09 12:41:21","video":"","vorDoi":"10.1007/s12668-024-01702-8","vorDoiUrl":"https://doi.org/10.1007/s12668-024-01702-8","workflowStages":[]},"version":"v1","identity":"rs-4216826","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4216826","identity":"rs-4216826","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}