Folate Dual-Targeted Amphiphilic Maltodextrin-Based

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Lactobionic/folate dual-targeted amphiphilic maltodextrin-based micelles for targeted co-delivery of sulfasalazine and resveratrol to hepatocellular carcinoma Doaa M. Anwar, Sherine Nabil Khattab, Maged W. Helmy, Mohamed K. Kamal, Adnan A. Bekhit, Kadria A. Elkhodairy, and Ahmed O Elzoghby Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00428 • Publication Date (Web): 15 Aug 2018 Downloaded from http://pubs.acs.org on August 16, 2018

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Bioconjugate Chemistry

Lactobionic/folate dual-targeted amphiphilic maltodextrin-based micelles for targeted codelivery of sulfasalazine and resveratrol to hepatocellular carcinoma Doaa M. Anwara,b, Sherine N. Khattaba,c *, Maged W. Helmya,d, Mohamed K. Kamale,f, Adnan A. Bekhita,g,h, Kadria A. Elkhodairya,b, Ahmed O. Elzoghbya,b,i,j * a

Cancer Nanotechnology Research Laboratory (CNRL), Faculty of Pharmacy, Alexandria University, Alexandria 21521, Egypt. b Department of Industrial Pharmacy, Faculty of Pharmacy, Alexandria University, Alexandria 21521, Egypt c Department of Chemistry, Faculty of Science, Alexandria University, Alexandria 21321, Egypt. d Department of Pharmacology and Toxicology, Faculty of Pharmacy, Damanhour University, Damanhour, Egypt e Department of Toxicology, Central labs of Alexandria, Health Affairs Directorate, Alexandria 21518, Egypt f Department of Oceanography, Faculty of Science, Alexandria University, Alexandria 21321, Egypt g Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Alexandria University, Alexandria 21521, Egypt h Pharmacy Program, Allied Health Department, College of Health Sciences, University of Bahrain, P.O. Box 32038, Kingdom of Bahrain i Division of Engineering in Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA (Current Address) j Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA 02139, USA (Current Address)

*Corresponding Authors. Prof. Sherine Khattab. E-mail Address: [email protected]; [email protected] Cell Phone: (002) 01223140924, Tel: (002) 035841866 Dr. Ahmed Elzoghby. E-mail Address: [email protected] ; [email protected] Cell Phone: (001) 781-366-8703, Tel: (001) 617-768-8994

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Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract In this study, promising approaches of dual-targeted micelles and drug-polymer conjugation were combined to enable injection of poorly soluble anti-cancer drugs together with site-specific drug release. Ursodeoxycholic acid (UDCA) as a hepatoprotective agent was grafted to maltodextrin (MD) via carbodiimide coupling to develop amphiphilic maltodextrin-ursodeoxycholic acid (MDCA)-based micelles. Sulfasalazine (SSZ), as a novel anti-cancer agent, was conjugated via a tumor-cleavable ester bond to MD backbone to obtain tumor-specific release whereas resveratrol (RSV) was physically entrapped within the hydrophobic micellar core. For maximal tumortargeting, both folic acid (FA) and lactobionic acid (LA) were coupled to the surface of micelles to obtain dual-targeted micelles. The decrease of critical micelle concentration (CMC) from 0.012 to 0.006 mg/ml declares the significance of dual hydrophobicized core of micelles by both UDCA and SSZ. The dual-targeted micelles showed a great hemocompatibility, as well as enhanced cytotoxicity and internalization into HepG-2 liver cancer cells via binding to over-expressed folate and asialoglycoprotein receptors. In vivo, the micelles demonstrated superior anti-tumor effects revealed as reduction in the liver/body weight ratio, inhibition of angiogenesis and enhanced apoptosis. Overall, combined strategies of dual active targeted micelles with bioresponsive drug conjugation could be utilized as a promising approach for tumor-targeted drug delivery. Keywords: Maltodextrin; Micelles; Sulfasalazine; Resveratrol, Dual targeting; Drug-polymer conjugate; Hepatocellular carcinoma. 1. Introduction Hepatocellular carcinoma (HCC) is considered the fifth most common cancer type that is responsible for hundreds of thousands annual mortalities. Moreover, it is the third main reason of 2


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death-related to cancer.1 A great attention has been particularly directed toward overcoming the problems related to the conventional chemotherapy using new delivery systems for the anticancer drugs.2 Using the nano-sized polymeric carriers for the delivery of chemotherapeutic agents has many benefits involving improved circulation times, more desirable bioavailability and use of low drug concentration which results in reduced side effects.3-4 High drug entrapment, site-specific release and improved tumor accumulation are the most important characteristics of successful anticancer drug-loaded nano-carriers. 5-6 Amongst nano-carriers with high drug loading, polymeric micelles have obtained growing attention due to their high loading potential of poorly water-soluble drugs into their internal hydrophobic core. 7-8 Micelles are commonly fabricated from amphiphilic co-polymers developed using synthetic hydrophilic and hydrophobic polymers. However, a great interest has been paid to the use of natural biopolymeric nano-carriers such as polysaccharides and proteins as efficient anticancer drug carriers.9-10 Recently, there is enormous concern in developing new amphiphilic polysaccharides which have the ability to form micelles via self-assembly. Maltodextrin (MD) is a hydrophilic polysaccharide produced by enzymatic hydrolysis of starch. 11 In this research, MD was chemically modified by ursodeoxycholic acid (UDCA), a water insoluble cholagogue, hepatoprotective and cholelitholytic agent.12 Due to the amphiphilic characteristics of this conjugate, MDCA can form micelles in water that can incorporate hydrophobic chemotherapeutic agents. Aside from a suitable drug loading, another pre-requisite for successful anti-cancer drug delivery is the site-specific drug release at tumor site maximize the anti-tumor efficacy and decrease the side effects. Therefore, in our study, we combine the benefits of both polymeric micelles and polymerdrug conjugates to enable injection of high loading hydrophobic anti-cancer drugs into the blood beside the drug release at specific site. Polymer-conjugated drugs generally exhibit several 3



advantages such as high stability, extended half-life, improved water solubility, as well as sitespecific drug release. 7 Finally, in order to accomplish the maximum accumulation of the drug into tumor cells, tumortargeted drug delivery can be achieved via decorating the surface of micelles with targeting ligands like peptides, hormones, antibodies, and small components such as folic acid (FA) and lactobionic acid (LA). 13-14 FA has been applied as a ligand in order to deliver the cytotoxic agents into the tumor tissue specifically, because of its teeny size, lack of immunogenicity, high availability, and simple coupling to other component. Folate receptors (FR) are widespread on many HCC cells. Meanwhile, normal tissue distribution of the FA receptor is very little, making it a perfect marker for the drug delivery targeting to HCC. 15 On another hand, LA has been used to effectively target human HCC cells over-expressing asialoglycoprotein receptors (ASGPR). Coupling of LA to etoposide (ETO)-loaded d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) nanocarriers has enhanced its internalization and cytotoxicity to HepG2 cells in addition to higher anti-tumor efficacy in HCC-bearing animals compared to non-targeted nanoparticles. 16 Dual targeting to the receptors, is a novel strategy for anti-cancer drug delivery, which can enhance the density of the available receptors for accumulation of the micelles into tumor cell and also can reduce the nonspecific uptake of micelles into normal cells. Therefore, in this study, the advantages of both FA and LA as a dual active targeting to HCC have been utilized by covalent attachment to the surface of MDCA micelles to selectively deliver anticancer drugs to liver cancer cells. Combination therapy of cancer is gaining growing attention, for preferable long-term efficacy and also to minimize the side effects. 17 Signaling pathways in diseased cells can be modulated by multi-agent therapy in order to increase the therapeutic effect and to overcome the mechanisms of resistance.18 4


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Sulfasalazine (SSZ) has been a mainstay in the treatment of Crohn’s disease and ulcerative colitis. Robe et al. reported lately, that SSZ provokes apoptosis in many types of cancers, such as lung carcinoma, esophageal cancer, and brain cancer.19 Recently, Guo et al.20 have reported that SSZ could suppress the growth of SK-Hep-1 and Huh-7 hepatocellular carcinoma cells through autophagy. On another hand, RSV, a naturally occurring polyphenolic phytoalexin, has drawn great attention due to its beneficial anti-cancer effects. RSV effectively inhibits cell proliferation through inducing cell cycle arrest in G1 as well as G2/M phases. Moreover, other anti-cancer mechanisms of RSV include induction of reactive oxygen species (ROS) generation, which can trigger autophagy as well as subsequent apoptotic cell death. 21 Based on these data, the synergistic combination of RSV with SSZ in treatment of liver cancer would be expected to enhance their anti-tumor efficacy. However, the clinical application of both RSV and SSZ was hampered by their poor aqueous solubility hindering their intravenous (i.v.) administration. Moreover, RSV has some drawbacks such as poor stability, and shorter biological half time,

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whereas SSZ may cause

gastrointestinal events, acute pancreatitis and diarrhea.23 Therefore, novel combined tumor-targeted delivery systems are needed to reduce the high dose of SSZ, minimize side effects, as well as enhance the therapeutic activity of both drugs in liver cancer. In this study, we suggest for the first time as far as we know, dual-targeted amphiphilic MDCA micelles for targeted co-delivery of SSZ and RSV for HCC therapy. First, amphiphilic co-polymer was synthesized by carbodiimide coupling of UDCA to maltodextrin backbone. Second, the hydrophobic drug, SSZ, was covalently-bonded to the MD fragment of the micelles via ester bond stable at systemic circulation but can be cleaved at tumor cells thus enabling its specific release at tumor sites and reducing its systemic toxicity. Third, RSV was physically incorporated into the 5



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hydrophobic core of micelles to overcome its high lipophilicity and enable its parenteral administration, providing sustained drug release pattern. Finally, as an active targeting approach to maximize drug accumulation in tumor cells, 24 the surface of micelles was dual-decorated with FA and LA to facilitate binding to FR and ASGPR thus enhancing internalization into HCC cells via receptor-mediated endocytosis. The developed micelles were investigated in vitro as well as in vivo in order to compare the anti-tumor efficacy of the combined drug-loaded micelles with the free drugs. 2. Results and discussion 2.1. Synthesis and characterization of MDCA conjugate (F0) This study was performed in order to evolve injectable micelles for delivery of poorly soluble anticancer medication. It describes the formulation of new amphiphilic MDCA micelles (F0) for codelivery of SSZ and RSV to HCC. In physiological conditions, the newly prepared amphiphilic conjugate could self-assemble into micelles composed of UDCA inner core which is considered as hydrophobic drug reservoir and hydrophilic MD shell to evade opsonisation. MDCA co-polymer was synthesized by conjugating UDCA to MD through the formation of an ester bond in presence DIC (N,N′-diisopropylcarbodiimide) and Oxyma as coupling reagent,

25

which enable acid

activation of UDCA to form an Oxyma active ester. Thus, the activated carboxyl group can undergo nucleophilic substitution by the hydroxyl group of MD in presence of DMAP (4dimethylaminopyridine) forming the ester bond, Scheme 1. 26 The conjugation reaction of MD with UDCA was confirmed by spectroscopic methods (FTIR, 1H NMR). The FTIR of MD shows a broad absorption band at the range 3400-3100 cm-1 corresponding to OH groups and absorption bands at 2927 cm-1 corresponding to the aliphatic sp3 C-H bonds. While the MDCA conjugate shows an absorption bands at 1707 and 1156, 1025 cm−1, 6


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corresponding to the C=O and the C-O groups of the formed ester bond. 27 The coupling was also confirmed by 1H-NMR (Fig. 1 A) where the spectrum of MDCA conjugate shows a doublet peak at δ 5.52 ppm corresponding to the anomeric protons of the MD skeleton. Furthermore, peaks in the range of 2.9 to 5.30 ppm correspond to (OH) group of both UDCA and MD, and also correspond to the (C-H) protons of MD. In addition, the 1H-NMR spectrum showed the singlet peak at 0.57 ppm corresponding to the CH3 group of UDCA, and a multiplet peak was observed in the range 0.83–2.50 ppm corresponding to the sp3 protons (alkane protons) of UDCA.

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The

number of molecules of UDCA conjugated to one molecule of MD was predicted from the ratio of integration of the doublet peak at δ 5.52 ppm corresponding to the anomeric protons of the MD skeleton to the integration of the singlet peak at 0.57 ppm corresponding to the CH3 group of UDCA. It was estimated that ~2 UDCA molecules were conjugated to each MD molecule. 2.2. Synthesis and characterization of SSZ-MDCA conjugate (F2) Sulfasalazine-MDCA conjugate (SSZ-MDCA) was prepared by the reaction of SSZ with MDCA co-polymer in presence of NHS and EDC.HCl through the formation of the desired ester bond,28,29,30 to inhibit its release into circulation and enable its release in tumor cells upon micellar degradation and bond cleavage by lysosomal enzymes. This would result in minimized side effects and enhanced accumulation of SSZ within tumor cells and hence maximized anti-tumor efficacy. The conjugation of carboxyl group of SSZ to the hydroxyl group of MDCA conjugate was confirmed by FTIR and 1H-NMR. The FTIR spectrum of SSZ-MDCA conjugate confirms the coupling of SSZ to the MDCA conjugate. Two characteristic absorption bands were observed at 1640 and 1414 cm−1 characteristic for the aromatic C=C bond of SSZ. In addition, two bands at 1372 and 1154 cm−1 corresponding to the SO2 group of SSZ were observed. In addition, the 1HNMR spectrum of SSZ-MDCA conjugate (Fig. 1 A) shows a multiplet (m) peak at 6.60-8.59 ppm, 7



characteristic of the aromatic protons of SSZ which confirms its conjugation with MDCA copolymer. 31 From the ratio of integration of the singlet peak at 0.57 ppm corresponding to the CH3 group of UDCA and the integration of the aromatic protons of SSZ, we can predict that ~1-2.5 molecules of the drug SSZ were conjugated to MDCA co-polymer. The conjugation efficiency (CE %) and total drug content of SSZ (wt. %) in the micelles was calculated indirectly by quantifying the un-conjugated drug or ligand during equilibrium dialysis method using UHPLC-MS/MS. The transition and confirmatory transition 398.900 to 380.600 and 398.833 to 223.700 was used for quantitation of SSZ. The conjugation percentage of SSZ was calculated to be 27.5% and the SSZ content (wt. %) was 13.8% (weight of SSZ for each 100 mg of co-polymer) (Table 1). The results were in a good agreement with the results obtained from the 1HNMR spectrum of SSZ-MDCA conjugate. 2.3. Synthesis and characterization of targeted SSZ-MDCA conjugate Actively-targeted nanocarriers are ideal platforms that enable nano-therapeutics to be targeted specifically to the cancer cell.32 Therefore, we decorated the surface of SSZ-MDCA micelles with two targeting ligands (FA and LA) aiming to enhance their internalization into liver cancer cells via binding to both FR and ASGPR over-expressed by HCC cells. Both targeting agents have been conjugated to the surface of SSZ-MDCA co-polymer through ester bond formation.30 The carboxylic acid group of FA was preactivated by using EDC.HCl/K salt of oxyma to form an intermediate active ester capable of covalent attachment to the hydroxyl group of MD.25,33,34 Although FA has two –COOH groups at α and γ positions which can act as suitable functional groups for covalent attachment, it was proved that the higher reactivity of the γ-COOH of FA makes it more prone to this reaction. 35 The success of FA coupling to SSZ-MDCA conjugate was confirmed by 1H-NMR and FTIR. The FTIR spectrum of FA-SSZ-MDCA (F3) shows a 8


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characteristic absorption band at 1700 cm-1 corresponding to ester C=O stretching vibration band. In addition, the most characteristic bands of FA at 1693, 1650 (amide I) and 1538 (amide II) provide evidence for FA conjugation. The bands at 3820, 3744, 3394 and 3417 cm−1 in the spectrum correspond to NH2 and secondary NH groups of FA and the NH group of SSZ. The 1H-NMR spectrum of FA-SSZ-MDCA conjugate (Fig. 1 B) shows peaks at the range δ 6.308.40 ppm corresponding to the aromatic protons of both SSZ and FA, in addition to the NH2 protons of FA, and NH protons of SSZ and FA. The 1H-NMR spectrum showed also peaks at δ 3.53-5.40 ppm, which include the CH2 protons of FA. From the ratio of integration of the peaks corresponding to FA aromatic protons and protons of the CH3 group of UDCA observed as a singlet peak at 0.57 ppm, it was estimated that ~1 FA molecule was conjugated to each MD molecule. 36 All these results were also confirmed by LC-MS/MS for indirect determination of the conjugation efficiency of FA (Table 1). LA conjugation was performed as given previously in FA conjugation. LA-SSZ-MDCA (F4) structure was confirmed my FTIR and 1H-NMR spectra. The FTIR spectrum shows an absorption band at 2930 cm−1, assigned to the CH2 stretching bands. The band at 1704 cm−1 corresponds to the ester C=O group, where the bands at 1154, 1080 and 1025 cm-1 correspond to the C–O–C vibration. The 1H-NMR spectrum (Fig. 1 B) shows an additional peak at the range of 2.07-2.16 ppm which confirms LA conjugation. The conjugation efficiency of LA was determined by LCMS/MS (Table 1). Dual-targeted conjugate FA-LA-SSZ-MDCA (F5) was prepared as previously mentioned in FA conjugation. The FTIR spectrum of the co-polymer shows a characteristic absorption at 1700 cm-1 corresponding to ester C=O stretching vibration band, where the bands at 1155, 1080 and 1024 cm1

correspond to the C–O–C vibration. In addition, an absorption band at 2930 cm−1, assigned to the 9



CH2 stretching bands was observed. Characteristic bands of FA at 1650 (amide I) and 1538 (amide II) were also observed. The 1H-NMR spectrum (Fig 1 B) shows an additional peak at the range of 2.10-2.14 ppm indicating the presence of LA. In addition, a multiplet (m) peak at 6.35-8.31 ppm, corresponding to the aromatic protons of SSZ and FA was observed. Based on 1H-NMR and LCMS/MS analysis, the average number of LA and FA molecules coupled to each MD was estimated to be ~ 0.5 each (Table 1). 2.4. Physicochemical characterization of RSV-loaded SSZ-MDCA micelles In contrast to SSZ, which was covalently conjugated to the MD backbone, RSV was physically loaded into the hydrophobic core of MDCA micelles via simple solvent evaporation method. RSV was 57.4 % entrapped into MDCA co-polymer. It is possible that there are abundant intermolecular interactions acting together between the drug and carrier substance during the process of drug-loading such as hydrophobic interaction, hydrogen bonding, van der Waal forces. All these forces could help stabilization of the micelles and also play a role in the efficient entrapment of RSV.37 Coupling of SSZ to the micellar structure was found to significantly reduce the EE% of RSV to the range of 15-30% (Table 2). This may be explained by the steric hindrance induced by incorporation of the hydrophobic SSZ which can compete with RSV for entrapment into the micellar core resulting in reduced RSV encapsulation. Besides drug loading, the size and surface charge of micelles may play a crucial role in determining their fate in the body. After entrapment of RSV into the micelles, the average particle size was slightly reduced from 249.5±3.6 to 245.2±6.1 nm (F1, Table 2). The reduction of micelle size may result from the enhanced hydrophobic interaction, caused by an increase in the amount of RSV entrapped into the core of MDCA micelles. In fact, with the increase of the hydrophobicity of copolymer, the micelles form a more tightly packed core. 38 After the conjugation of SSZ, the particle size of micelles was slightly 10


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increased to 252.1±2.5 nm. Moreover, the addition of targeting ligands onto the micelles surface was found to increase its size to be in the range of 255.2±4.3 and 276.8±6.6 for F3 and F5, respectively (Fig. 2 A). The reasonable particle size of micelles (around 250 nm) and also their hydrophilic shell would be expected to cause prolonged circulation of these micelles resulting in their passive accumulation into tumor cells via the EPR effect. The zeta potential is a key indicator of the colloidal stability of micelles (Fig. 2 B). It is noteworthy to mention that the zeta potential of the fabricated micelles was decreased as we increased the substitution on the hydrophilic backbone from -24.2 to -13.7 mV, for F0 and F3, respectively. The relatively low negative charge on the surface has the advantage of preventing opsonization. The negative zeta potential of the fabricated micelles is in agreement with the results reported by Raveendran et al. 39 who fabricated curcumindextran micelles with negative zeta potential of −15.2 mV. Thus, the new micelles developed here, with appropriate size and negative surface charge could enhance circulation half-lives, evade the reticuloendothelial system (RES) and hence lead to enhanced targeting to liver cancer. RSV and SSZ in their natural state exist in crystalline form, which is characterized by the melting peaks around 265 and 258°C, respectively (Fig. 2 C). Our finding revealed the absence of RSV and SSZ endothermic peaks in the thermograms of micelles that indicated amorphization of the drugs due to entrapment of the first drug and conjugation of the second one within the fabricated dual-targeted micelles. 2.5. Morphological analysis The prepared F5 micelles appeared in spherical shape in the TEM micrograph, having a diameter in the range of 150 nm with no aggregation, referring to the relatively high stability of micelles. Moreover, the core-shell structure observed in the TEM micrograph gave an indication for the formation of the hydrophilic MD corona surrounding UDCA-SSZ hydrophobic core (Fig. 2 D). It 11



was found that the diameter of micelles determined by TEM was much smaller than the diameter of micelles measured by dynamic light scattering (about 276.8 nm) which determines the diameter of micelles with the solvation layers. The decrease in size determined by TEM may be due to the shrinkage that occur during the drying process of sample preparation for the TEM experiment. 5 2.6. Critical micelle concentration (CMC) The CMC of both MDCA and dual-targeted FA/LA-SSZ-MDCA micelles was determined using pyrene, as it is considered a non-aqueous fluorescent probe. At low co-polymer concentrations, pyrene showed low fluorescence intensity in hydrophilic medium because of its hydrophobic characteristics and it’s self-quenching. Accordingly, no significant change in the total fluorescence intensity was recorded. The fluorescence intensity was markedly increased when the co-polymer concentration increased. This may be attributed to the partitioning of pyrene molecules into the hydrophobic core of the self-assembled micelles (Fig. 3 (A, B)). From the plot of intensity ratio (I343/I334) of pyrene emission versus log c of both MDCA and FA-LA-SSZ-MDCA micelles, the CMC values were found to be about 0.012 and 0.006 mg/ml, respectively (Fig. 3 (C, D)). The reduction of CMC value may refer to the increase in the hydrophobicity of the micelles core due to the conjugation of SSZ as a hydrophobic drug. This reslt is one of the essential parameters for the stability of micelles in systemic circulation upon dilution by the large blood volume. 40 2.7. In vitro drug release In vitro release profiles of free RSV vs RSV release from MDCA (F0) and FA-LA-SSZ-MDCA (F5) micelles were compared using dialysis bag method. As shown in Fig. 4 A, free RSV was nearly released completely after 2 h. While, the release profile of RSV entrapped in the micelles was biphasic and showed fast release during the first 4-6 h of about 51.0% ± 3.8 followed by very slow release with about 42.4% ± 5.1 of RSV released for the remaining 24 h. The initial burst 12


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release may be due to some of the RSV that is localized in the shell or at the core-shell interface, while the slow release of the drug in the second phase could be due to the amount of the drug entrapped physically within the non-aqueous core of the micelles. 41 The slow drug release from the fabricated micelles will allow the usage of the drug in parenteral delivery, where long circulation of the carriers may lead to increase drug accumulation at the site of solid tumors as well as localize drug release.42 Concerning SSZ release, the release was done at a different pH (7.4, 5.0, and 4.0). No drug was released from the micelles over the entire period of in vitro release test at pH 7.4 when injected to UHPLC-MS/MS, while at pH 5.0, a very small amount of SSZ (about 2.0%) was released throughout the period of test. On the other hand, at more acidic pH 4.0, the release of SSZ was increased gradually until reaching 24.69% after 3 days (Fig. 4 B). No release was observed at pH of 7.4 may be attributed to the formation of a stable ester bond that may resist the premature release of SSZ in the physiological pH after i.v. administration of the prepared micelles thus decreasing the systemic side effects. Instead, it can be released specifically at the tumor sites after the cleavage of the bond by the effect of the acidic medium (pH 4.0) as well as lysosomal enzymes. 2.8. Physical stability and redispersibility After storing at 4°C for three months, the physical stability of all micelles was evaluated in terms of change in the particle size and zeta potential with time (Fig. 4 (C, D)). The FA/LA-dual targeted micelles showed particle size of 251.5±5.9 and 326.3±7.8 nm, respectively after three months of storage, compared to the initially stored (255.2±5.1 and 294.4±4.5 nm) micelles. Also, there is no dramatic change in the zeta potential values after three months compared to the fresh ones. This reflect the stability of FA/LA-targeted micelles. These results agree with the reported high stability 13



of the dispersed solutions of amphiphilic cholic acid‐modified-dextran sulfate micelles. 43 On the contrary, dual-targeted micelles demonstrated lower stability during the test period showing gradual size increase with time. The size of micelles was increased from 276.8±6.6 nm to 306.2±9.4 nm after eight weeks of storage and kept increasing till reaching 366.3±5.3 nm after three months. It is well known that micelles are dynamic systems that have the ability to reaggregate than other solid polymeric nanoparticles. The conversion of the prepared micelles to dry powder by lyophilization was reported to increase the physical stability of micelles. 44 In our study, without a need of a cryoprotectant, a fluffy powder was obtained, that can form a colloidal solution when dispersed again in water without any aggregation. This may be attributed to the presence of MD which is considered as a lyoprotectant which protects the particles during freeze-drying preventing their aggregation. 45 Mosquera et al. 46 added MD to the borojó pulp before freezedrying for the sake of improving the stability of borojó powders, reducing the powder hygroscopicity and increasing its glass transition temperature (Tg). The reconstituted lyophilized FA/LA-RSV-SSZ-MDCA F5 micelles demonstrated PS of 278.8±9.6 and 255.5±8.4 nm before and after lyophilization respectively, with redispersibility index of 0.91, where a value lower than 2.0 is considered efficient.47 Moreover, there is no dramatic change in zeta potential of the micelles obtained after freeze-drying (Table 3). 2.9. In vitro hemolysis and serum stability Serum protein adsorption onto polymeric micelles in human blood serum is a major stability challenge for the use of micelles for parenteral drug delivery. When incubated with serum, unstable micelles rapidly adsorb these proteins and as a result form aggregates.48 The PS of FA/LA-RSV-SSZ-MDCA micelles was increased from 276.8±6.6 to 290.1±8.2 nm after 1 h incubation with 10% FBS (Fig. 5 A). After 5 h, the PS of micelles reached 378.4±3.8 nm whereas 14


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the PS was decreased again to 283.1±8.2 nm after 6 h incubation. The initial elevation in the PS may be attributed to the slight adsorption of BSA onto the surface of micelles and formation of protein corona around polymeric micelles. Meanwhile, the recurrent reduction in the PS could be due to the osmotic pressure resulted from serum proteins causing water to escape from the micelles core and subsequent shrinking of the micelles.26-49 Moreover, the ability of the prepared micelles to cause lysis in rat erythrocytes was evaluated. The leakage of hemoglobin from RBCs was used to quantitatively compare the membrane-damaging properties of micelles. As shown in Fig. 5 (B, C), the FA/LA-RSV-SSZ-MDCA micelles demonstrated only 0.73% hemolysis less than the accepted nontoxic level (2%), according to American Society for Testing and Materials (ASTMF 756-00, 2000). 50 These results owe to the negative zeta-potential on the surface of the prepared micelles that make it compatible with RBCs. Rabeendran et al. reported the hemolysis percentage of curcumin-loaded dextran micelles to be 0.06 ±0.01 % which complies with our results. 39 This is an indication for the safety of the prepared micelles which could reveal the importance of using MD in the formulation of nano-carriers as being non-toxic, non-immunogenic, biodegradable, biocompatible and does not cause lysis for RBCs. 2.10. In vitro cytotoxicity The anticancer efficacy of the free and combined RSV/SSZ solution was compared to the developed micelles against human liver cancer HepG-2 cells at 24 h (Fig. 5 D). The IC50 of free drugs compared to the micelles was summarized in Table 4. The reduction in the IC50 of RSV/SSZ combination solution as compared to the free SSZ and free RSV on HepG-2 gave us an idea about the synergism between the two drugs. The very low IC50 of RSV compared to that of SSZ is beneficial to decrease the IC50 of SSZ in the drug combination. As a result, the treatment dose of 15



SSZ can be reduced and hence its side effects are diminished. The results of the IC50 of the free drug combination showed that the IC50 of SSZ decreased in comparison with that of free SSZ. Regarding the micelles, it seemed that the targeted ones (F3, F4 and F5) enhanced the combination potency to show IC50 values with 1.22, 1.31, and 1.49 folds reduction compared to the free combined drug solution (p < 0.05) and 1.14-fold reduction for the non-targeted micelles (F2). The dual-targeted micelles F5 showed the lowest IC50 value compared with other prepared micelles. The reliability of study was ensured using CompuSyn software (version 1) described by Chou and Talalay,51 where the Combination Index (CI) and Dose Reduction Index (DRI) were used to compare between the different formulations and the free combination. The obtained results ensured the synergistic effect of combining SZZ and RSV as their free combination showed CI lower than 1 (0.962). Moreover, the obtained results showed the same pattern of superiority of different formulations than the free combination, especially F4 & F5, where their combination indices CIs were 0.852 and 0.794, respectively, which indicate that these two formulations succeeded to achieve a synergy between SZZ and RSV. Moreover, the DRIs of SZZ were 1.74 and 1.97 in F4 and F5 respectively. While, the DRIs of RSV were 2.14 and 2.45 in F4 and F5, respectively as shown in Fig. 6 A and Table 5. The ability of FA and LA to internalize efficiently into HepG-2 cancer cells by membrane-bound FR and ASGPR receptor-mediated endocytosis could explain the elevation in the cytotoxicity of the dual-targeted micelles, which accordingly increases the accumulation of drugs into the tumor cells leading to improved anti-tumor activity. 52 These results clearly demonstrate the effectiveness of our targeting ligands. Moreover, the results confirmed that blank MDCA micelles had no obvious adverse effect on cell viability, so we can exclude the possible effect of carrier on cell viability. 2.11. In vitro cellular-uptake and flow cytometry 16


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In order to make fluorescent labeling for the prepared micelles, the hydrophobic dye coumarin-6 was entrapped into the core of the micelles. The uptake of both dual-targeted and non-targeted coumarin-6 labeled MDCA micelles versus free coumarin-6 solution after 4 h and 24 h incubation with HepG-2 cells at 37ºC was determined by confocal microscopy. Results demonstrated that both coumarin-6 labeled micelles exhibited clearly higher green fluorescence compared with free coumarin-6 which could be primarily assigned to better membrane internalization as a result of reduced particle size and decoration with targeting ligands. In order to ensure the reliability of results, flow cytometry analysis was performed to compare the endocytosis of FA-LA-dual targeted micelles and non-targeted micelles using HepG-2 cell line. When HepG-2 cells were incubated with dual targeted micelles, much higher cellular uptake was observed by comparing the fluorescent intensity with the non-targeted micelles as shown in Fig. 6 B. The geometric mean fluorescent intensities of F2 after 4 h (A1), F2 after 24 h (A2), F5 after 4 h (A3), and F5 after 24 h (A4) were 88.64, 364.21, 403.86 and 1970.02 respectively. It is worth mentioning that dualtargeted MDCA micelles showed more pronounced uptake than non-targeted MDCA ones (Fig. 7 (A, B, C)). This enhanced uptake could be explained by the targeting effect of FA and LA through their attachment to the overexpressed FR and ASGPR on HepG-2 cells through receptor-mediated endocytosis (RME) as the main mechanism which confirms the results of cytotoxicity. 53 2.12. In vivo anti-tumor efficacy The in vivo antitumor efficacy of the prepared micelles was investigated on HCC bearing mice. After the treatment period, the liver-to-body weight ratios were significantly (p

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