Camptothecin encapsulated into functionalized MCM-41: In vitro release study, cytotoxicity and kinetics

Accepted Manuscript Camptothecin encapsulated into functionalized MCM-41: In vitro release study, cytotoxicity and kinetics

Priyanka Solanki, Shweta Patel, Ranjitsinh Devkar, Anjali Patel PII: DOI: Reference:

S0928-4931(18)31774-0 https://doi.org/10.1016/j.msec.2019.01.065 MSC 9323

To appear in:

Materials Science & Engineering C

Received date: Revised date: Accepted date:

22 June 2018 4 January 2019 15 January 2019

Please cite this article as: Priyanka Solanki, Shweta Patel, Ranjitsinh Devkar, Anjali Patel , Camptothecin encapsulated into functionalized MCM-41: In vitro release study, cytotoxicity and kinetics. Msc (2018), https://doi.org/10.1016/j.msec.2019.01.065

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ACCEPTED MANUSCRIPT Camptothecin encapsulated into functionalized MCM-41: In vitro release study, cytotoxicity and kinetics Priyanka Solankia, Shweta Patelb, Ranjitsinh Devkarb and Anjali Patela*

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Vadodara 390002, Gujarat, India

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Department of Chemistry, Faculty of Science, The Maharaja Sayajirao University of Baroda,

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Department of Zoology, Faculty of Science, The Maharaja Sayajirao University of Baroda,

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Vadodara 390002, Gujarat, India

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*E-mail: [email protected]

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Abstract

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The application of MCM-41 functionalized by 12-tungstophosphoric acid (TPA) as drug delivery carrier for cancer treatment was studied by loading of Camptothecin (CPT). In-vitro controlled

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release study of CPT in Simulated Body Fluid (pH 7.4, 37 oC) was carried out under stirring as well as static conditions. The systems were also evaluated on cancer cells (HepG2) and the

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carriers are found to be non-toxic to the cancer cells. In order to see the influence of inorganic

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moiety on release rate of drug, study was also carried out with CPT loaded unfunctionalized MCM-41. A detailed study on release kinetics and release mechanism using First Order Release Kinetic Model, Higuchi Model, Koresmeyer-Pepps Model and Extended kinetic model was also carried out. Keywords: MCM-41; 12-tungstophosphoric acid; Camptothecin; in vitro release; MTT assay; kinetics

ACCEPTED MANUSCRIPT 1. Introduction Camptothecin (CPT) is a naturally occurring quinolone alkaloid which shows significant anticancer activity with a broad spectrum of human malignancies and CPT is an inhibitor of the DNA-replicating enzyme topoisomerase-I [1]. Unfortunately, the clinical application of CPT is

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hindered by its poor pharmaceutical profile, with extreme aqueous insolubility, low stability of

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the lactone form at physiological pH, and severe systemic toxicities which included myelosuppression, vomiting, diarrhoea, and hemorrhagic cystitis [2-4]. A better understanding of

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mode of action, chemistry and pharmacology of the CPT led to the development of water-soluble derivatives such as irinotecan, topotecan and 9-aminocamptothecin [5]. Although less active than

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the CPT [6], these derivatives have gained approval by the Food and Drug Administration (FDA)

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for treating cancers. However, these CPT derivatives still suffer from important drawbacks mainly related to the poor stability of the lactone ring, the short half-life of the compounds in

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blood and a number of non-resolved toxic effects. Therefore, the development of controlled

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delivery strategies could lead to significant advantages in the clinical use of these drugs. In this sense, CPT has been encapsulated in different vehicles, like PLGA microspheres [7], solid lipid

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nanoparticles [8], liposomes [9] and micelles [10]. Reports are also available for Camptothecin

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loading and release using functionalized as well as non-functionalized Silica nanoparticles [11] and polymeric nanoparticles [12]. Further, to modulate the release rate of CPT, carriers are functionalized by various organic molecules such as silica nanoparticles functionalized by 3Mercaptopropl group [13] and Nucleic acids [14]. Functionalization leads to overcome the mentioned problems faced by CPT. A literature survey shows that the functionalization was carried out using organic moiety only. It also shows that no reports are found on release study of CPT using either non-

ACCEPTED MANUSCRIPT functionalized MCM-41 or functionalized MCM-41. In 1992, Mobil Corporation have synthesized the mesoporous silica materials and called as MCM-41. These are amorphous inorganic materials consisting of silicon and oxygen in their framework. Its pore sizes are in range of 2-50 nm in dimension. This material has been widely used as carrier as having unique

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characteristic such as Ordered porosity at the mesoscale, variable pore size, high specific surface

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area, High adsorption capacity, High concentration of surface Si-OH groups through which it can

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interact with different functional group of drug. Hence, it was thought of interest to use combination of an inorganic moiety as a functionalizing agent and MCM-41 as new drug

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delivery systems. The idea was further motivated by recently published result by our group [15]

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where we have reported functionalisation of MCM-41 (pore diameter: 3.7 nm) by an inorganic moiety, 12-tungstophosphoric acids (TPA) and its use for the in vitro release of L-arginine where

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we found the beneficial effect of TPA and that encourage us to continue the work.

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So, as an extension of our work, for the first time, we describes use of MCM-41 with

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different pore diameter (4.9 nm) as carrier for CPT. The present article describes functionalization of MCM-41 (pore diameter: 4.9 nm) by TPA, encapsulation of CPT and

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characterization using various physicochemical techniques. For finding the cytotoxic effect of

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TPA, MTT-assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) of all the materials were carried out using HepG2 cancer cells line. In vitro release study of CPT, loaded with functionalized and unfunctionalized MCM-41 was carried out in SBF at pH 7.4 and temperature 37 0C under stirring as well as static conditions. Further, to see the effect of TPA on release rate of CPT, release profiles of drug loaded, functionalized and unfunctionalized MCM41 are compared. To investigate drug release kinetic and mechanism, different models such as

ACCEPTED MANUSCRIPT First ordered release kinetic Model, Higuchi Model, Korsmeyer-Peppas Model and Extended Kinetic Model were used. 2. Experimental

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2.1 Materials

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All chemicals used were of A.R. grade. Camptothecin (Sigma Aldrich),

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Cetyltriethylammoniumbromide (CTAB) (Lobachemie, Mumbai), Tetraethylorthosilicate

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(TEOS, Merck), NaOH (Merck), n-hexane (Merck), Mesitylene (Merck), 12-tungstophosphoric acid (Merck), Dimethyl sulphoxide (DMSO, Merck) and Liq. NH3 (Merck) were used as

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received. Human hepatocellular liver carcinoma (HepG2) cells were obtained from National

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Centre for Cell Science, Pune, India

Simulated body fluid (SBF): NaCl (7.996 g), NaHCO3 (0.350 g), KCl (0.224 g),

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K2HPO4.3H2O (0.22 g), MgCl2.6H2O (0.305 g), 1 molL-1 HCl (40 mL), CaCl2 (0.278 g), Na2SO4

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(0.071 g) and NH2C(CH2OH)3 (6.057 g) were dissolve in small amount of water and then dilute

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to 1 L with distilled water.

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2.2 Synthesis of MCM-41

MCM-41 was synthesized using reported procedure [16] with modification. Surfactant (CTAB, 4.38 g) and NaOH (1.2 g) were dissolved in 200 mL double distilled water. When the solution became homogeneous, TEOS (20.8 g) was added quickly with stirring. After 5 min, mesitylene (8.64 g) and hexane (3.1 g) was added to the stirred mixture. The resulting thick mixture was stirred vigorously for 10 min and then heated at 85 °C for 2 days with stirring. The resulting

ACCEPTED MANUSCRIPT product was filtered, washed with double distilled water, dried at 100 °C temperature. The obtained material was calcined at 550 °C in air for 5 h and designated as MCM-41. 2.3 Functionalization of MCM-41 using 12-Tungstophosphoric acid (TPA)

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MCM-41 was functionalized using TPA as functionalizing agent by incipient wet impregnation

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method. 30 % of TPA anchored to MCM-41 was synthesized. One g of MCM-41 was

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impregnated with an aqueous solution of TPA (0.3/30 g/mL of distilled water) and dried at 100 o

2.4 Encapsulation of CPT into TPA-MCM-41

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C for 10 h. The obtained material was designated as TPA-MCM-41.

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Encapsulation of CPT was carried out by soaking method. 221 mg of TPA-MCM-41 was soaked

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in 10 mL solution of CPT (22.1 mg/mL) in DMSO for 24 h with stirring in sealed glass vials. After 24 h, the mixture was filtered and washed with 10 mL of DMSO. The filtrate was analyzed

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for CPT contained using UV-Visible spectrophotometer at 370 nm. The obtained material was

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air dried and designated as CPT/TPA-MCM-41. CPT was loaded on MCM-41 following the same procedure and obtained material was designated as CPT/MCM-41. Drug loading amount

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was obtained by analyzing filtrate by UV-Visible spectrophotometer (Table 1) which is also

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confirmed by TGA analysis. 2.5 Leaching Test

Whether TPA is truly act as anchoring agent or not, leaching of TPA was investigated. TPA can be qualitatively characterized by the formation of heteropoly blue colour, when treated with a mild reducing agent such as ascorbic acid [17]. In the present study, this method was used for determining the leaching of TPA from the carrier. 1 g of TPA-MCM-41 was stir in 10 mL waterethanol mixture (40:60) for 24 h. Then 1mL of the supernatant solution was treated with 10%

ACCEPTED MANUSCRIPT ascorbic acid. Development of blue colour was not observed, indicating absence of any leaching. The same procedure was repeated with SBF, in order to check the presence of any leached TPA. The absence of blue colour indicated no leaching of TPA. 2.5 Characterizations

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TGA analysis was done on Mettler Toledo Star SW 7.01 from 30 oC to 500 oC at heating rate of

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10 oC/min in presence of nitrogen atmosphere. The FT-IR spectra of materials were obtained by using KBr palate on Perkin Elmer instrument. Adsorption-desorption isotherms of all

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synthesized materials were recorded on a Micromeritics ASAP 2010 Surface area analyzer at liquid nitrogen temperature. From the adsorption desorption isotherms, specific surface area was

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calculated using BET method. For activation, all materials were degassed at 90 oC for 6 hrs

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under 3-4 µm Hg. TEM analysis was carried out on JEOL (JAPAN) TEM instrument (modelJEM 100CX II) with accelerating voltage of 200 kV. The samples were dispersed in ethanol and

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ultrasonicated for 5-10 min. A small drop of the sample was then taken in a carbon coated copper

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grid and dried before viewing. The XRD pattern was obtained by using PHILIPS PW-1830. The conditions were: Cu Kα radiation (1.54 Ao), scanning angle from 0o to 10o. UV-spectral analysis

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of materials has been carried out using Perkin Elmer Lambda 35.

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2.6 Cytotoxicity study (i) In Vitro Study:

Human hepatocellular liver carcinoma (HepG2) cells were incubated at 37 °C with 5% CO2 in a water jacketed CO2 incubator (Thermo Scientific, Forma series II 3111, USA). Cells were seeded (1 × 105 cells) in a T25 flask and cultured in DMEM containing 10% FBS and 1% antibiotic−antimycotic solution. Cells were trypsinized every third day by subculturing with TPVG solution.

ACCEPTED MANUSCRIPT (ii) Cytotoxicity Assay: Cytotoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells (7 × 103 cells/well) were seeded in 96-well culture plates for 24 h and then treated with 0.1, 0.3 and 0.5 mg/mL concentrations of MCM-41, TPA-MCM-41, CPT/TPA-

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MCM-41 for 24 h. The doses of CPT treatment were 5, 15 and 25 μg/mL that were proportionate

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to the amount of CPT released from 0.1, 0.3 and 0.5 mg/mL CPT/TPA-MCM-41 respectively.

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orate to the Later on, 10 μL of MTT (5 mg/mL) was added followed by incubation for 4 h at 37 °C. Formazan formed at the end of the reaction was dissolved in 150 μL of DMSO and

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absorbance was read at 540 nm using an ELX800 Universal Microplate Reader (Bio-Tek

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instruments, Inc., Winooski, VT) and the percentage cytotoxicity was calculated [18].

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2.7 In vitro release study

In vitro release of CPT was carried out by soaking drug loaded samples in 100 mL of SBF at 200

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rpm at 37 oC temperature under stirring as well as static condition. At proper time interval, 1 mL

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of release fluid was taken and fresh SBF was added to maintain the constant volume of the system. The 1 mL fraction was diluted and analyzed for CPT contains using UV-Visible

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spectrophotometry at 370 nm. All the experiments were repeated three times. For comparison of

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both systems, the same concentration of drug was taken for release study (7.1 × 10-3 mg/mL). 3. Result and Discussion 3.1 Characterizations Fig.1 shows TGA curve of all the materials. TGA curve of TPA exhibits weight loss in three stages at 100, 200 and 485 oC. These can be attributed to initial weight loss due to adsorbed

ACCEPTED MANUSCRIPT water, second weight loss due to loss of water of crystallization and last may be attributed to the decomposition of the Keggin structure of TPA into the simple oxides [19]. TGA curve of MCM-41 shows initial weight loss of 6.14 % at 100 oC which may be due to the loss of adsorbed water. The final 7.92 % weight loss above 450 oC may be due to the

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condensation of silanol groups to form siloxane bonds. The TGA of TPA-MCM-41 (Fig. 1c) shows initial weight loss of 3.6 % due to the loss of adsorbed water. Second weight loss of 1.2 %

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between 150 and 250 oC corresponds to the loss of water of crystallization of Keggin ion. Further

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a gradual weight loss was also observed from 250 to 500 oC due to the difficulty in removal of

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water contained in TPA molecules inside the channels of MCM-41 [20]. The TGA curve of CPT/MCM-41 and CPT/TPA-MCM-41 shows initial weight loss of 42% and

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19% due to the loss of adsorbed water up to 150 oC. Further weight loss of 7.3% and 4.9% from

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200 to 550 oC, may be due to the removal of CPT. The calculated amount of CPT from TGA was found to be 49±2 mg and 71±2 mg of CPT for MCM-41 and TPA-MCM-41 (Table 1)

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respectively, in good agreement with that obtained by UV-Visible analysis. The loading

𝑑𝑟𝑢𝑔 𝑒𝑛𝑐𝑎𝑝𝑠𝑢𝑙𝑎𝑡𝑒𝑑 𝑐𝑎𝑟𝑟𝑖𝑒𝑟 𝑤𝑒𝑖𝑔ℎ𝑡

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𝐿𝐸(%) = (

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efficiency/capacity has been calculated using equation (1) and shown in Table 1. ) ∗ 100

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Fig. 1. TGA curve of (A) TPA, (b) MCM-41, (c) TPA-MCM-41, (d) CPT/MCM-41 and (d)

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CPT/TPA-MCM-41

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Fig. 2 Shows powder XRD of MCM-41, TPA-MCM-41, CPT/MCM-41 and CPT/TPA-

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MCM-41. The low angle XRD of MCM-41 displays an intense diffraction peak at 2θ = 2° which are assigned to the lattice faces (100), suggesting a hexagonal symmetry of MCM-41 (Figure 2.).

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In addition to this weak peaks are observed at 2θ = 3-5° with very low intensity which are

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sometimes difficult to observed due to instrumental error [21]. Further, Lu et al have reported similar pattern of XRD peaks of MCM-41 [22]. It is well known that the low angle XRD pattern

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are sensitive to pore filling and loaded materials show lowered intensity of characteristic peak compared to pure one and this reflect in the XRD pattern of TPA-MCM-41, CPT/MCM-41 and CPT/TPA-MCM-41 which shows characteristics peak at 2θ = 2° with lower intensity. This also suggests the insertion of CPT into the mesoporous channels of carrier which is in good agreement with reported results [23, 24]. It is interesting to observe that the rest of the peaks are disappearing especially in case of CPT/TPA-MCM-41. This is because of further loading of CPT into functionalized MCM-41 may block the channels which have been further confirmed by BET

ACCEPTED MANUSCRIPT analysis. Further the hexagonal mesoporous structure of all the materials are confirmed by TEM

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(Fig.3).

Fig. 2. Powder XRD of (A) MCM-41, (b) TPA-MCM-41, (c) CPT/MCM-41 and (d) CPT/TPA-

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MCM-41

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Fig.3. shows TEM images of MCM-41, CPT/MCM-41, TPA-MCM-41 and CPT/TPAMCM-41. TEM images of all show hexagonal and very well ordered porous structure. In addition, well dispersion of CPT is observed in case of CPT/MCM-41. TEM images of TPAMCM-41 shows absence of crystalline phase of TPA inside the channels of MCM-41 indicating high dispersion of TPA and absence of agglomeration. CPT/TPA-MCM-41 shows ordered porous structure with absence of any agglomeration indicating the well dispersion of CPT into TPA-MCM-41.

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Fig. 3. TEM images of (a) MCM-41, (b) TPA-MCM-41, (c)CPT/MCM-41 and (d) CPT/TPAMCM-41 at 100 nm magnification

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Fig. 4. shows Nitrogen adsorption-desorption isotherm of MCM-41, TPA-MCM-41,

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CPT/MCM-41 and CPT/TPA-MCM-41 and textural properties of these are shown in Table 1. The all four systems shows type-IV isotherm according to the IUPAC classification with H1 hysteresis loop which is characteristic of mesoporous materials and suggest the intact structure of MCM-41. In case of CPT/MCM-41 (Table 1), surface area and pore volume are decreases which suggest the insertion of CPT into the mesoporous channel of MCM-41. Similarly, Decrees in all parameters in CPT/TPA-MCM-41 as compared to that of TPA-MCM-41 indicates the insertion of CPT into the TPA-MCM-41.

ACCEPTED MANUSCRIPT The higher loading of CPT in case of MCM-41 compared to TPA-MCM-41 may be due to the presence of less available space in case of TPA-MCM-41 as mesoporous channels of MCM-41

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are already occupied by TPA moiety.

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Fig. 4. Nitrogen adsorption-desorption isotherm MCM-41, TPA-MCM-41, CPT/MCM-41 and CPT/TPA-MCM-41

ACCEPTED MANUSCRIPT Table 1 Textural properties of materials, amount of drug loading and loading efficiency

1.19 0.55

Amount of drug encapsulated (mg/g of material) 71±2

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CPT/TPA-MCM41

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49±2

Loading Efficiency/Loading Capacity 7.1±2

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Pore volume (cm3/g)

MCM-41 CPT/MCM-41

Specific surface area (m2/g ) 890 502

4.9±2

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Materials

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Fig. 5 shows FTIR spectra of MCM-41, CPT/MCM-41, TPA-MCM-41 and CPT/TPAMCM-41. FTIR spectrum of MCM-41 (Fig.5a) shows broad band at 1100–1300 cm-1, 3448 cm-1

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corresponds to asymmetric stretching vibration of Si–O–Si and symmetric stretching vibration of

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Si–OH group, respectively. The bands at 801 cm-1 and 498 cm-1 represent the symmetric stretching and bending vibration of Si–O–Si. The reported bands of CPT are at 3425 cm-1, 1736

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cm-1, 1648 cm-1, and 1436 cm-1 corresponding to OH stretching, ester (carbonyl) stretch,

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carbonyl stretching and C-N stretching vibration, respectively [25]. The FTIR spectrum of CPT/MCM-41 (Fig.5b) shows entire bands related to MCM-41 indicating intact structure of

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MCM-41 and CPT. It also shows bands shifting from 1736 to 1874 cm-1 corresponding to Ester

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(Carbonyl) which indicates the interaction of CPT with MCM-41 through ester carbonyl group. The reported bands for TPA, at 1088, 987, and 800 cm-1 corresponding to W–O–W bending, W–O and P–O symmetric stretching, respectively, are absent in TPA-MCM-41 (Fig.5c). If TPA is dispersed onto the surface of MCM-41, the mentioned bands for TPA should be seen in the FT-IR spectra. The absence of respective FTIR bands of TPA in TPA-MCM-41 may due to the overlapping of TPA bands with that of MCM-41. All the bands of MCM-41 were observed in TPA-MCM-41 which confirms the intact structure of MCM-41. The FTIR spectrum

ACCEPTED MANUSCRIPT of CPT/TPA-MCM-41 shows all the bands related to TPA-MCM-41 and CPT. It also shows shifting in band corresponding to ester carbonyl, from 1736 cm-1 to 1884 cm-1 which indicate the

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interaction of CPT with TPA-MCM-41 through C=O group.

MCM-41

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Fig. 5. FTIR spectra of (a) MCM-41, (b) CPT/MCM-41, (c) TPA-MCM-41 and (d) CPT/TPA-

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Fig. 6. shows UV absorption spectra of MCM-41, TPA-MCM-41 and CPT/TPA-MCM-41. MCM-41 does not show any absorption band. TPA-MCM-41 show bands 253 nm which is corresponding to W-O d-d charge transfer. CPT/TPA-MCM41 also shows band at 249 nm, along with this it shows band 370 nm which is corresponding to CPT moiety.

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Fig. 6. UV absorption spectra of MCM-41, TPA-MCM-41 and CPT/TPA-MCM-41 3.2 Evaluation of in vitro cytotoxicity-MTT assay

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Induction of apoptosis in cancer cells leading to their termination has been extensively reported

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by use of several anti-cancer agents including Camptothecin (CPT). Inhibition of topoisomerase

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followed by cytochrome C release and mitochondrial hyperpolarization is reported to be induced by CPT in mammalian cancer cells [26]. Mitochondrial dysfunction is assessed by MTT assay to

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establish anticancer potential of test compounds. In the present study, Human hepatocellular liver

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carcinoma (HepG2) cells were treated with MCM-41, TPA-MCM-41, CPT/TPA-MCM-41 or CPT and MTT assay was performed. Metabolically active cells produce a purple coloured formazan at the end of the assay and the intensity of colour reflects upon the functional status of mitochondria [18, 27]. MCM-41 showed ≤ 10% cytotoxicity in the said doses (0.1, 0.3, 0.5 mg/mL, Fig. 7). The functionalized material, TPA-MCM-41 recorded < 30% cytotoxicity whereas functionalized carrier loaded with drug CPT (CPT/TPA-MCM-41) showed > 40% at 0.5 mg/mL dose. These observations are of relevance because CPT/TPA-MCM-41 (at 0.5 mg/mL

ACCEPTED MANUSCRIPT where in 25µg/mL CPT was released) accounted for 9.2% higher cytotoxicity than CPT treated group at the same dose. Overall, the results indicate that the TPA-MCM-41 carrier is non-toxic to the cells but the drug loaded carrier accounts for highest percentage of cytotoxicity amongst all groups. These results imply towards CPT/TPA-MCM-41 mediated improved delivery of

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CPT that can be of significance in cancer therapy.

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Fig. 7. Effect of materials on the cytotoxicity of HepG2 cells. Control cells did not show cytotoxicity. Results are expressed as mean ± SD for n=3. $$P < 0.01 and $$$P < 0.001 as

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compared to control of respective groups and #P < 0.05, ###P < 0.001 as compared to respective

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concentrations of MCM-41. **P < 0.01 and ***P < 0.001 as compared to respective

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concentrations of TPA-MCM-41.

ACCEPTED MANUSCRIPT 3.3 In vitro release study 3.3.1 Effect of Stirring To evaluate the effect of stirring on release rate of CPT, release study was carried out under

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stirring and static condition and results are shown in Fig.8. It is observed that under static

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condition slower release profile is obtained for both systems i.e CPT/MCM-41 and CPT/TPA-

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MCM-41. Under static condition, intially, 18% and 10% of CPT is release which is reached to 33% and 27% up to 10 h from MCM-41 and TPA-MCM-41, respectively. However,

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comparatively fast release profile is obtained under stirring condition for both systems. Slower

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diffusion of CPT molecules, under static condition could be the Reason of slower release of

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CPT.

Fig. 8. In vitro release profile of CPT/MCM-41 and CPT/TPA-MCM-41 under (a) stirring and (b) static condition 3.3.2. Effect of TPA on release rate To see the influence of TPA on release rate of CPT, release profile of CPT/MCM-41 and CPT/TPA-MCM-41 was compared and results are shown in Fig. 9. Initially, 28% and 21% of

ACCEPTED MANUSCRIPT CPT is released and reached to 72% and 62% up to 10 h from MCM-41 and TPA-MCM-41, respectively. It reached to 98% and 90% up to 30 h for MCM-41 and TPA-MCM-41, respectively. Here, slower release profile is obtained for CPT/TPA-MCM-41 as compared to CPT/MCM-41.

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This is may be because of the more attractive interaction between the CPT molecules and TPA-

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MCM-41. As stated earlier, TPA has terminal free oxygen through which it can bind with drug.

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This is may be the reasons of slower release of CPT from TPA-MCM-41.

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Fig. 9. Comparison of Release profile of CPT/MCM-41 and CPT/TPA-MCM-41

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3.4 Release kinetics and mechanism To investigate drug release kinetic and mechanism, The CPT release data were fitted with First ordered release Kinetic Model, Higuchi Model [28-30]. The type of diffusion of CPT in support as well as the relation between the surface properties and release rate was studied by KorsmeyerPeppas Model (KPM) and Extended Kinetic Model (EKM) [31, 32]. (i)

First order release kinetic model

ACCEPTED MANUSCRIPT First order release kinetic model is used to study the dissolution of drug encapsulated in porous matrices. According to this model, rate of release is concentration dependent. Fig. 10 shows first ordered release kinetic model of CPT/MCM-41 and CPT/TPA-MCM-41, where log of % remaining data are plotted against time in hr. The First order release kinetic model was best fitted

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with higher linearity and higher co-relation coefficient (R2= 0.9775) for CPT/TPA-MCM-41.

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This suggests the, CPT release is concentration dependent process and more ordered release is

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obtained for CPT/TPA-MCM-41 system.

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Fig. 10. First order release kinetic model of CPT/MCM-41 and CPT/TPA-MCM-41 Higuchi model

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The Higuchi model (Fig. 11) describes the percentage release versus square root of time dependent process based on fickian diffusion. According to this model release mechanism of drug involves simultaneous penetration of SBF into the pores, dissolution of drug molecule and diffusion of these molecules from the pores. The release mechanism of CPT is best explained by this model with high linearity and high correlation coefficient (R2 = 0.997) for CPT/TPA-MCM41. This suggests that release of CPT follows fickian diffusion mechanism as well as more ordered release profile was obtained in case of CPT/TPA-MCM-41.

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Korsmeyer-Peppas Model

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Fig. 11. Higuchi model of CPT/MCM-41 and CPT/TPA-MCM-41

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Korsmeyer and Peppas (1984) developed an empirical equation to analyze both Fickian and nonFickian release of drug. Mt/M∞ = Ktn. n is the empiric exponent which indicates type of release

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mechanism, where n = 0, 1.0 and 0.5 indicates Zero-Order, First-order and Higuchi model

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respectively [18]. In the present case, the found value of n is 0.48 and 0.5 for CPT/MCM-41 and CPT/TPA-MCM-41 respectively (Fig.12), confirming that the present systems follow Higuchi

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model. For KPM, higher linearity and correlation Co-efficient (R2) was obtained for CPT/TPA-

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MCM-41.

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Extended Kinetic Model

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(iv)

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Fig. 12. KPM model for CPT/MCM-41 and CPT/TPA-MCM-41

EKM model described the drug concentration dynamics in the bulk liquid, on solid and at the

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interface. It includes the diffusion steps for the drug transport in pores and in the external liquid

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film (surrounding the carrier) to the bulk liquid. Due to the concentration gradients, the drug follows diffusion path in the pores and external liquid film. The estimated kinetic parameters of

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EKM model are shown in Table 2 and EKM prediction of the drug concentration dynamics in the

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bulk liquid, on solid and at the interface are displayed in Fig. 13 for CPT/MCM-41 and CPT/TPA-MCM-41. On comparing the estimated desorption-adsorption equilibrium constants K

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= k2/k1 from Table 2, it is clear that release tendency is higher for CPT/MCM-41 (K =6.19) compared to CPT/TPA-MCM-41 (K = 4.76). This may be due to the strong interaction of CPT with terminal oxygen of TPA which can hold the CPT molecules for longer time.

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Concentration dynamics

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Fig. 13. Experimental data and predictions of CPT release from MCM-41 and TPA-MCM-41 in

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Table 2

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SBF by Extended Kinetic Model.

Estimated parameters of KPM and EKM model for CPT release from MCM-41 and TPAMCM-41. Units: k1, k2 (h-1 mL mg-1); K= k2/k1 Materials CPT/MCM-41 CPT/TPA-MCM-41

Model Korsmeyer-peppas KPM: k1= 0.0061 K= 6.19 k1= 0.0105 K= 4.76

Extended EKM: n = 0.48 n = 0.5

ACCEPTED MANUSCRIPT Conclusion The present contribution reports the release study of CPT from MCM-41 functionalized by an inorganic moiety, 12-tungstophophoric acid. The MTT study shows that MCM-41 and TPAMCM-41 are non-toxic. However, CPT/MCM-41 and CPT/TPA-MCM-41 show toxicity

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towards the HepG2 cell which is as expected and thus can further be used for in-vivo studies.

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Kinetic and mechanistic study shows that CPT release follows the first order release kinetic

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model and Higuchi model. This is further supported by KPM. The value of EKM equilibrium constants (K) for CPT/TPA-MCM-41 is lower as compared to CPT/MCM-41.This is because of

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TPA, which holds the drug molecules for longer time and help in obtaining controlled and

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delayed release profile. Acknowledgments

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PS is also thankful to University Grant commission (UGC), New Delhi for the award of Senior

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Research Fellowship. We are also thankful to Department of Chemistry, The Maharaja Sayajirao University of Baroda, for nitrogen adsorption-desorption analysis and TGA analysis. Authors are

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Reference

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also thankful to Mr. Ali Asagar Vohra for MTT analaysis.

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ACCEPTED MANUSCRIPT Camptothecin encapsulated into functionalized MCM-41: In vitro release study, cytotoxicity and kinetics Priyanka Solankia, Shweta Patelb, Ranjitsinh Devkarb and Anjali Patela*

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Vadodara 390002, Gujarat, India

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Department of Chemistry, Faculty of Science, The Maharaja Sayajirao University of Baroda,

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Department of Zoology, Faculty of Science, The Maharaja Sayajirao University of Baroda,

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Vadodara 390002, Gujarat, India

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*E-mail: [email protected]

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Graphical abstract

In vitro controlled release of Camptothecin from functionalized MCM-41, Kinetics as well as MTT study was carried out. MTT study shows that drug loaded materials are cytotoxic to the HepG2 cancer cells.

ACCEPTED MANUSCRIPT Highlights Encapsulation of Camptothecin into functionalized and unfunctionalized MCM-41.

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Characterization of drug loaded and unloaded materials using various techniques.

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In vitro cytotoxic assay of all materials by MTT study using HepG2 cancer cells.

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Effect of stirring on release rate and Comparison of release profile of all materials.

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Investigation of drug release kinetic and mechanism using various models.

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