TiO2 nanocomposites: Antibacterial and antitumor activities

Journal Pre-proof Synthesis and Design of Norfloxacin drug delivery system based on PLA/TiO2 nanocomposites: Antibacterial and antitumor activities Nehal Salahuddin, Mohamed Abdelwahab, Mohamed Gaber, Sahar Elneanaey PII:

S0928-4931(19)31374-8

DOI:

https://doi.org/10.1016/j.msec.2019.110337

Reference:

MSC 110337

To appear in:

Materials Science & Engineering C

Received Date: 12 April 2019 Revised Date:

23 September 2019

Accepted Date: 16 October 2019

Please cite this article as: N. Salahuddin, M. Abdelwahab, M. Gaber, S. Elneanaey, Synthesis and Design of Norfloxacin drug delivery system based on PLA/TiO2 nanocomposites: Antibacterial and antitumor activities, Materials Science & Engineering C (2019), doi: https://doi.org/10.1016/ j.msec.2019.110337. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

Synthesis and Design of Norfloxacin Drug Delivery System Based on PLA/TiO2 Nanocomposites: Antibacterial and Antitumor Activities Nehal Salahuddin*, Mohamed Abdelwahab, Mohamed Gaber, Sahar Elneanaey Chemistry Department, Faculty of Science, Tanta University, Tanta, 31527 Egypt

TOC Figure

Synthesis and Design of Norfloxacin Drug Delivery System Based on PLA/TiO2 Nanocomposites: Antibacterial and Antitumor Activities Nehal Salahuddin*, Mohamed Abdelwahab, Mohamed Gaber, Sahar Elneanaey Chemistry Department, Faculty of Science, Tanta University, Tanta, 31527 Egypt

Abstract Biodegradable, biocompatible and non-toxic polymer-based nanoparticles are the novel nanotherapeutic tool which is used for adsorption and encapsulation drugs. Extended release formulation of Norfloxacin antibiotic, chemotherapeutic agent model, drug in the form of encapsulated and loaded poly (lactic acid) nanocomposites-based Titanium dioxide (PLA/TiO2) was developed. Nanocomposites were prepared using different contents (1, 3, 5 wt %) and morphologies of TiO2 (spheres (S), rods (R). The dispersion of TiO2 was aided by ultrasonic technique followed by solution casting method. The morphology, particle size, crystallite size and composition of the nanocomposites were examined by SEM, TEM, XRD and FTIR. The crystallinity and thermal behavior of the nanocomposites were characterized by DSC and TGA. NOR was loaded onto TiO2 nanospheres (NOR@TiO2 (S)) and the optimum conditions for loading was investigated. Pseudosecond order model was the more adequate to represent the kinetic data. The equilibrium data followed Freundlich adsorption isotherm and the adsorption process was exothermic. NOR@TiO2 (S) was encapsulated into PLA and in vitro release behavior of drug was compared with NOR adsorbed into PLA (NOR@PLA) and nanocomposites (NOR@PLA/TiO2) using different pH (6.7, 7.4) media. To study the mechanism of NOR release, first order, Higuchi, Hixon Crowell and KorsmeyerPeppas models were applied on the experimental results. The cytotoxicity of the loaded nanocomposites using MMT assay was studied against HePG-2, MCF-7, HCT-116, PC-3, Hela, WI-38 and WISH cells. The encapsulated (NOR@ 5S/En 1

PLA) showed the highest cytotoxic efficacy with moderate effect on normal cells. Moreover, the nanocomposites have great potential against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Salmonella and Klebsiella pneumonia. NOR@ (PLA/TiO2) nanocomposites showed better antibacterial efficacy than NOR encapsulated nanocomposites. The nanocomposites could be effective vehicles for the sustained delivery of toxic anticancer drug. Keywords: Poly (lactic acid); Nanocomposites; Norfloxacin; Extended release; Titanium dioxide. *Corresponding author: Nehal Salahuddin Email: [email protected] 1. Introduction Fluoroquinolone, Norfloxacin is an antibacterial agent with broad spectrum activity against Gram negative and some Gram positive aerobic bacteria [1]. It is used as antibiotic for treatment Urinary tract infections, Prostatitis, Genital tract infections, Gonorrhea and eyes infections which caused by E.coli, V.choleae, Salmonella, Shigella and Campylobacter [2]. It is administrated through oral route with only 30-40% oral bioavailability and short half-life time (3-4 h) in serum and plasma [3]. To improve the patient compliance, many researchers developed an extended release formulation to reduce the schedule of administration, improve bioavailability and decrease the fluctuation that was observed with twice daily dosing of immediate release. Hydroxypropyl methyl cellulose (HPMC) and sodium alginate were worked synergistically and provided prolonged release up to 12 h at the upper part of gastrointestinal tract in the floating tablets to overcome the multiple dosing required in

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the treatment due to the short half-life of NOR in plasma [4]. Thahera et al. developed an extended release formulations of NOR up to 24h using intermediate molecular weight poly(ethylene oxide) [5]. Utilizing guar gum, sodium CMC, HPMC and NaHCO3 as a gas generating agent in direct compression method, extended the drug release over a period of 7-12 h [6]. Proniosomes of NOR was prepared to sustain the release of NOR throughout 24 h [7]. Dave et al. developed lipid polymer hybrid nanoparticles for topical and site targeting delivery of NOR with 90% cumulative drug release in 24 h [8]. NOR acts as inhibitor for type II topoisomerase enzyme that present in prokaryotic and eukaryotic cells [9]. Tentative efforts have been made to shift from antibacterial to antitumor activity. Recently, Fluoroquinolone, Ciprofloxacin, Ofloxacin, Voreloxin have shown inhibition the growth in several cancer cell lines [10]. Voreloxin is currently being evaluated in a phase 2 clinical trial for ovarian cancer treatment [11]. A real challenge is currently addressed in the development of drug delivery system that has the ability to target the site of infection without serious side effects, and the main goal of cancer therapeutics is to increase the survival time by reducing the systemic toxicity of chemotherapy. Poly (Lactic acid) (PLA) has eminent advantages including good biocompatibility, biodegradability and stellar mechanical properties that creates this polymer common in the biomedical applications such as bone screws, surgical sutures, controlled drug delivery system and tissue engineering [12]. Applying PLA as a carrier for drugs enhanced the consumed ratio of drugs and alleviates the damage to kidney and liver transpired due to taking medicine several times a day to preserve the drug at a certain level. PLA is relatively hydrophobic polyester that can undergo chain disruption in human body and degrade into nontoxic by-product, lactic acid; CO2 and H2O which then discarded through the Krebs cycle and in the urine. PLA exhibited high efficacy in

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encapsulation protein, oridonin, hormones, psychotic drugs and restenosis drugs [13]. Slow drug release formulation of PLA was developed for Mitomycin and Dexamethasone sodium phosphate, [14], Haemoglobin, Lidocaine and Vapreolide [15]. TiO2 nanoparticles have a special merit due to good chemical stability, good biocompatibility, low toxicity and photocatalytic efficacy with antitumor efficiency [16]. TiO2 have been used as carrier for chemotherapeutic agents such as Doxorubicin, Temozolomide, Camptothecin and Danuorubicin [17-21] with high effectiveness than free drugs. Mesoporous Titania nanoparticles have a high affinity to phosphate species such as DNA and can be used as bioimaging by inclusion fluorescent molecule [22]. In addition, the photo catalytic activity of TiO2 helps in reducing drug resistance and provide cancer cell targeting [23]. TiO2 exists in three forms, Anatase type, Rutile type and Brookite type. In general, Anatase form is preferred due to high photocatalytic activity as a consequence of high negative conduction band edge potential [24]. The performance of TiO2 depends on its morphology [25], one dimensional nano structural materials such as nanotubes have superior properties including catalytic, optical, mechanical and biological activities [26]. TiO2 bioactive nanoparticles are incorporated into PLA matrices to enhance cell attachment and proliferation on the composite surface [27]. Mixing of TiO2 nanoparticles with PLA directly, leads to their agglomeration within PLA matrices. These aggregations can decrease the interfacial areas between the polymer chains and TiO2 leading to poor properties. To improve the dispersion of TiO2 into PLA matrices, L-Lactic acid oligomers were grafted onto the surface of TiO2 nanoparticles by polymerization of LLactic acid using stannous octanoate as a catalyst [28]. Nakayama et al. modified TiO2 using propionic acid and n-hexylamine to disperse them into PLA matrices uniformly [29] however, photodegradability of nanocomposites was efficiently modified. In

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another publication, incorporation of TiO2 into PLA/PU leads to enhancement of impact toughness of the blend without bioactivity under simulated physiological conditions [30]. Interestingly, bacteriostatic activity of TiO2/PLA nanocomposites irradiated by sun light was greatly increased with only 3% TiO2 in comparison with PLA. This good bacteriostatic activity creates the possibility of using these nanocomposites in biomedical applications as drug delivery and antibacterial materials [31]. Processing procedures have significant influence on the distribution of TiO2 nanoparticles in PLA matrix [32]. PLA/TiO2 nanocomposites were prepared using different methods including grafting method [33], Van extruder technique [34], spin coating technique [35] to investigate the mechanical, thermal and electrical behavior for humidity sensing applications. To the best of our knowledge, there is not enough literature available on using ultrasonication technique for producing PLA/TiO2 nanocomposites and their utilization in drug delivery system. In the present work, we have synthesized TiO2 nanomaterials via two different routes, hydrothermal and sol gel method. PLA/TiO2 nanocomposites have been prepared using ultra sonication technique. The sonication is beneficial for the dispersion of TiO2 into polymer matrix. In this study, NOR was loaded into TiO2 (S) nanoparticles followed by encapsulation onto PLA. The drug loading capacity was optimized and compared with the NOR adsorption into the nanocomposites so that an extended release formulation can be obtained. The kinetics of drug release were investigated using different pH media. The efficacy of the nanomedicine as antibacterial and anticancer agent was studied. 2. Materials and methods 2.1. Chemicals Titanium (IV) Isopropoxide (TTIP) was supplied by Kiahida Chemical Japan, isopropanol ((CH3)2 CHOH), nitric acid (HNO3), ethanol (C2H5OH), sodium 5

hydroxide (NaOH), hydrochloric acid (HCl), chloroform (CHCl3), dimethyl sulfoxide (DMSO), Roswell Park Memorial Institute (RPMI)-1640 medium and 3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide dye (MTT) were purchased from Sigma-Aldrich Company, St. Louis, USA. A commercial PLA (Mw=4032D) was obtained from Nature works® Co, LLC, USA. Norfloxacin (NOR) drug was purchased from Epico, Egypt. Fetal bovine serum was purchased from GIBCO, UK. Cancer cell lines were obtained from ATCC via Holding company for biological products and vaccines (VACSERA), Cairo, Egypt. 2.2. Methods 2.2.1.

Synthesis of TiO2 Nanospheres (S)

The synthesis of TiO2 nanospheres, TiO2 (S), follows the procedure described by Mahshid et al.[36] . First, a suspension (a) was prepared by adding 10 ml TTIP to 30 ml Isopropanol, A solution (b) was prepared by adding drops of nitric acid (HNO3) to 500 ml distilled water until the pH changed to acidic (pH=2). The solution (b) was added slowly to the suspension (a) and mixed together under high stirring until gel was achieved. The gel was continuously stirred at 70oC for 20h. The final precipitate was collected and washed with distilled water and ethanol followed by drying in oven at 100oC for 2h and calcinated at 600oC for 2h to afford TiO2 (S), yield=2.5g. Hydrolysis of metal alkoxide transpired through SN1 mechanism assisted by H+ followed by dehydration of Ti(OH)4 to TiO2 in condensation step [37]. 2.2.2. Preparation of TiO2 Nanorods (R) Teflon-lined autoclave was used to prepare TiO2 nanorods, TiO2 (R), by modification of TiO2 (S). Accordingly, 1g of TiO2 (S) was added to 40 ml NaOH (10M) in 100 ml autoclave and heated for 48 h at 200ºC. Then, the precipitate was filtered, washed by 6

0.1M HCL solution and rinsed with deionized water several times until the pH changed to be 7±0.5. Then, the final precipitate was dried in oven at 60ºC for 12 h [38]. 2.2.3. Preparation of PLA/TiO2 (S, R) Nanocomposites PLA pellets were dried at 60ºC for 6 h. PLA/TiO2 nanocomposites were prepared by solution casting method. Accordingly, 0.99 g of PLA was dissolved in chloroform by stirring for 1-2 h till complete dissolution. 0.01g of TiO2(S) was added to PLA, and then the components were sonicated for 60 minutes followed by stirring for 48 h. Then, the suspension was dropwise into ethanol, the precipitate (PLA/1S) was filtered and dried in oven at 40ºC for 12h. To obtain different ratios and morphologies of TiO2, the same method was done using 0.97 and 0.95 g of PLA with 0.03 and 0.05g of TiO2 (S), respectively to afford PLA/3S, PLA/5S. In addition, 0.99, 0.97 and 0.95 g of PLA with 0.01, 0.03 and 0.05 g of TiO2 (R) to obtain PLA/1R, PLA/3R, PLA/5R, respectively. 2.2.4.

Preparation of Norfloxacin drug solution

Stock solution of Norfloxacin drug was prepared by dissolving 0.01g of drug in 5 ml dimethyl sulfoxide, then completed to 250 ml with water in measuring flask to obtain 40 ppm. Experimental drug solutions of different concentrations were prepared by dilution of drug stock solution. Absorbance of these concentrations were determined by UV-Visible spectrophotometer at λmax = 272 nm and calibration curve is drawn. 2.2.5.

Adsorption

of

Norfloxacin

nanocomposites

7

drug

onto

PLA

and

PLA/TiO2

Adsorption process of NOR was carried out by adding 0.01 g of PLA/TiO2 using different morphologies (S, R) and different contents of TiO2 to 10 ml of drug solution (14 ppm) in dark bottles and kept under shaking condition for 6 h at 400 rpm, centrifuged and the loading efficacy was monitored by measuring the concentration of residual drug in the supernatant using UV-visible spectrophotometer at λmax =272 nm. The precipitates (NOR@PLA/1S, NOR@PLA/3S, NOR@PLA/5S, NOR@PLA/1R, NOR@PLA/3R, NOR@PLA/5R) were centrifuged and dried in oven at 40ºC for 12h. NOR Loading Efficiency (LE) (%) was calculated from equation (1):

Loading Efficiency (%) =

2.2.6.

mass of NOR loaded on carrier × 100 (1) mass of carrier loaded with NOR

Loading of Norfloxacin drug onto TiO2 (S) nanoparticles

Loading was carried out by immersing 0.01, 0.03 and 0.05 g of TiO2(S) into 10 ml of drug solution (14 ppm) in dark bottles, sonicated for 30 min and kept under shaking at 400 rpm. The equilibrium was attained after 300 min, 180 min, and 90 min, respectively. Then, the supernatant was withdrawn to calculate the drug concentration and the precipitate was collected to afford NOR@1S, NOR@3S and NOR@5S with loading percentages 84.02±1.73%, 90.96±1.84% and 94.33±0.5%, respectively. An important observation was noticed about the stability of the drug during loading into anatase TiO2 (S). A peak shift for the drug was observed upon using TiO2 (R). Anatase phase of TiO2 showed less reduction tendency than other phases of TiO2 [37]. NOR Loading Efficiency (LE) (%) was calculated from equation (1) and adsorption isotherms models as Langmuir isotherm (2) and Freundlich isotherm (3) were applied on the loading of NOR onto TiO2 (S) nanoparticles [39].

8

!

"!

=

1 ! + (2) #. "%&' "%&'

1 log "! = log *+ + log ,

! (3)

Where, qe is drug concentration loaded at equilibrium, Ce is drug concentration in solution at equilibrium, qmax, is the amount required to form a monolayer (mg/g); KL is Langmuir equilibrium constant, Kf and n are the Freundlich constants. Adsorption kinetic models as Pseudo-first order (4) and Pseudo-second order (5) are used to explain the mechanism of loading NOR onto TiO2 (S) nanoparticles [40].

log(q 5 − q 7 ) = log q 5 −

K9 t (4) 2.303

1 1 t = + t (5) : q7 K:q 5 q5 qt is the amounts of drug adsorbed at time t, K1, K2 are the rate constants of pseudofirst order and pseudo-second order, respectively. 2.2.7.

Encapsulation of TiO2 (S) nanoparticles loaded NOR into PLA

Encapsulation process of NOR@TiO2 (S) into PLA was carried out by dispersing NOR@1S, NOR@ 3S and NOR@ 5S in 0.99, 0.97 and 0.95 g of PLA dissolved in 20 ml chloroform respectively, followed by sonication for 20 min, stirring for 48 h, and pouring the mixture in petri dish till the solvent completely evaporated to obtain NOR@1S/En PLA, NOR@3S/En PLA and NOR@5S/En PLA nanocomposites films, respectively. 2.2.8.

Release Study

9

Phosphate buffer solution was prepared by mixing 0.1M of (NaH2PO4) and 0.1M of (Na2HPO4) and the pH was adjusted by 1M HCL and 2M NaOH to be 6.7 and 7.4. 0.01 g of NOR@S,R/PLA powders with different TiO2 ratios and 0.25 g of NOR@ S/En PLA films were stirred at 200 rpm in 10 ml of buffer solution in different pHs at 37 ºC. The release of drug was followed by measuring the absorbance on UV-Visible spectrophotometer and the concentration was calculated from calibration curve of the drug. Buffer solution with different pHs was replaced by fresh buffer solution periodically. The release percentage was calculated according to equation (6), the release was analyzed using pseudo-first order (7), Higuchi (8), Hixon-Crowell (9) and Korsmeyer-Peppas (10) models [41].

Release % of NOR =

amount of NOR in buffer solution × 100 (6) amount of NOR loaded on carriers

log(100 − >) = log 100 − ?9 @ (7) > = ?B @9/: D

(8) D

(100 − >)E = 100E − ?BF @

(9)

GH ⁄GI = *@ K

(10)

Where, W is the cumulative drug release percent during time t., Mt/Mα is the fraction of drug release, K1, KH, KHC, K are first order rate constant, Higuchi drug release constant, Hixson-Crowell drug release rate constant and constant includes structural and characteristics of the dosage form respectively, n is release component which emphasized the mechanism of drug release.

2.2.10. Antibacterial activity test

10

The in vitro antibacterial activity of carriers before and after loading Norfloxacin drug were tested against different bacterial strains including Staphylococcus aurous, Pseudomonas aeruginosa, Salmonella, Klebsiella pneumonia and Escherichia coli by agar diffusion method [42]. Sterilized nutrient agar was prepared, spread into sterilized plates, allowed to solidify and wells were made. The bacteria were revived by inoculating in broth media and grown at 37 oC for 24 h. The wells were filled with all carriers before and after NOR loading. All the plates were incubated at 37 oC for 24 h. The diameters of inhibition zones were recorded and compared with NOR drug [43]. 2.2.11. Cytotoxicity assay and antitumor evaluation MMT assay [44] was used to detect cytotoxic effects of drug loaded nanocarriers on different tumor and normal cell lines. Cell lines include hepatocellular carcinoma separated from liver, HepG 2 (ATCC®HB-8065); colorectal carcinoma separated from colon, HCT 116 (ATTC®CCL-247); ductal carcinoma separated from mammary gland breast, MCF-7 (ATCC®CRL-3435); adenocarcinoma separated from prostate, PC-3 (ATTC®CRL-1435); epitheliod adenocarcinoma separated from cervix, Hela (ATCC®CCL-2); human amnion membrane, WISH (ATCC®CCL-25); human lung fibroblast, WI-38 (ATCC®CCL-75)) were tested, Doxorubicin drug was used as a standard cytotoxic agent for comparison. Cells were batch cultured in fresh RPMI-1640 medium with 10% fetal bovine serum then, a mixture of 100 unit’s/ml penicillin and 100µg/ml streptomycin antibiotics was added at 37oC in a 5% CO2 incubator. The cells were inoculated in a 96-hole plate at a density of 1.0x104 cells for each hole for 48 h. After incubation period, the cells were treated with various concentrations of samples. After 24 h of drug treatment, 20 µl of MTT solution (5 mg/ml) was added and incubated for 4 h. DMSO (100 µl) is added to all holes to 11

dissolve the purple formazan formed. The colorimetric evaluation is measured using a plate reader (EXL 800, USA) to determine absorbance at 570 nm, and then the relative cell viability (%) was established. 2.2.12. Instruments The crystallinity and crystallite size of nanoparticles and nanocomposites were verified by X-ray diffraction (XRD) (GNR, APD 2000). The phase composition of TiO2 (S) was calculated from integrated intensities of the anatase (101), rutile (110) and brookite (111) peaks by equation (12) [45]:

>L =

ML (12) 0.886M& + ML

Where, Wr is the weight fraction of rutile phase, Ar is the integrated intensity of rutile (110) peak; Aa is the integrated intensity of anatase (101) peak. From XRD pattern, the crystallite size of TiO2 (S, R) was calculated by Debye-Scherer's formula given by equation (13) [46]:

O=

?. P (13) Q. RST U

D is the crystallite size, λ is the wavelength of the X-ray radiation (0.15406 nm), β is the line width at half maximum height, θ is the diffraction angle and K equal 0.94. Nanoparticles and nanocomposites morphology were examined with Scanning Electron Microscope (JEOL SEM, JSM-6510LV-Japan) and Transmission Electron Microscope JEOL, JEM-2100- Japan. Fourier transform infrared (FTIR) spectroscopic analysis of PLA, nanoparticles and nanocomposites were done using JASCO FT-IR 4100, Japan. The samples were pelletized with KBr disk. Thermogravimetric analysis (TGA) and

12

Differential scanning calorimeter (DSC) were performed on Perkin Elmer STA. Nitrogen gas was the purging gas and the heating rate was 10oC/min in the range of 25-800 oC. The degree of crystallinity (Xc) was calculated from DSC curves using Eq. 14: ∆B

Y VW = (9Z∅)∆B ∗ V 100

(14)

Y

where ∅ is the weight fraction of the dispersed phase in the composites, ∆]% is the melting enthalpy (J/g) that was calculated from the fusion peak in the DSC curve and ∗ ∆]% is the heat of fusion for completely crystallized PLA (93.1 J/g).

The adsorption and release of drug were followed by measuring the absorbance of NOR on UV-Visible Spectrophotometer (UV-2101 PC) using quartz cuvette and the concentration was calculated from calibration curve of the drug. Drug solutions were separated from the samples by centrifuge using JANETZKI T5. The pH of buffer solutions was measured by 3510 bench JENWAY pH Meter.

3.

Results and Discussion

TiO2 nanomaterials were synthesized via two different routes, Sol-Gel and Hydrothermal methods to obtain spherical (S) and rod (R) nanoparticles, respectively. PLA/TiO2 nanocomposites were prepared by solution casting method. NOR was loaded into TiO2 (S) and the optimum condition for high loading was investigated. The TiO2 loaded NOR was encapsulated into PLA. The release, antibacterial and anticancer efficacy of NOR adsorbed onto PLA/TiO2 nanocomposites and NOR loaded TiO2 encapsulated PLA were studied. 3.1. Characterization of carriers The XRD pattern of TiO2 (S) nanoparticles synthesized by sol gel method (Fig. 1A) showed peaks lying at 2θ= 25.2o, 37.8o, 47.9o, 53.8o, 62.6o, 68.7o, 75.8o characteristic 13

of 101, 103, 200, 105, 213, 116 and 107, respectively of anatase phase according to JCPDS file 00-001-0562. In addition, peaks at 31o and 70.1o corresponding to 211 and 332 of Brookite phase according to JCPDS file 00-002-0514, Peak at 27.6o corresponding to 110 of Rutile phase according to JCPDS 00-001-1292 with only 14% of rutile phase as calculated using eq. 12. XRD pattern of TiO2 (R) nanoparticles synthesized by hydrothermal method (Fig. 1A) showed peaks at 25.6o, 36.5o, 40.1o, 49.5o, 57.6o, 60.5o, 65.2o characteristic of 111, 102, 202, 312, 231, 600 and 123 planes, respectively for Brookite phase with good agreement with JCPDS files 00002-0514. It was reported that complete conversion of anatase phase to rutile phase was obtained at 800oC with spherical morphology [47]. The crystallite size calculated using eq. 13, for TiO2 (S) is 18 nm and for TiO2 (R) is 20 nm. The XRD pattern of PLA (Fig.1B) showed sharp peaks at 2θ= 17.05o and 19.30o, these peaks are assigned to α-phase crystallite of PLA which is orthorhombic with chains in helical conformation [48]. The XRD patterns of PLA containing 1, 3, 5 wt% TiO2 nanoparticles with different morphology are shown in Fig. 1B, C. The peak characteristic to TiO2 (S) present at 2θ=25.2o is observed in PLA/3S, PLA/5S with crystallite sizes ~17 nm. The peaks characteristic to TiO2 (R) present at 2θ=25.6o was not clearly shown and peak at 2θ=31o characteristic of brookite is observed in PLA/3R, PLA/5R with crystallite sizes ~23nm, ~27nm, respectively. No new peaks were observed for nanocomposites implying absence of chemical bonds [48]. The XRD patterns of NOR@TiO2 (S) and NOR@ S/En PLA nanocomposites are shown in (Fig. 1D), It is observed that loading NOR on TiO2 nanoparticles don't affect crystallinity and size of TiO2 and the peak characteristic to TiO2 (S) present at 25.2o is observed in NOR@ 3S/En PLA and NOR@ 5S/En PLA with crystallite size of

14

~19nm, ~29 nm, respectively. There is no shift in the PLA peaks indicating that, the crystallinity did not change by addition of TiO2 (S) nanoparticles loaded drug. FTIR spectra of TiO2 (S, R), PLA, PLA/TiO2 Nanocomposites, NOR and NOR encapsulated nanocomposites are shown in Fig.2. TiO2 nanoparticles spectra show characteristic peaks at 3414 cm-1 (-OH stretching), 1628 cm-1 (-OH bending) which are due to reabsorption of water molecules from the ambient conditions [49]. Absorption peaks at 668 cm-1 in TiO2(S), 748 cm-1 in TiO2 (R) are due to υ(Ti-O-Ti) stretch peak [50]. In PLA spectrum, there is a peak at 3449 cm-1 due to –OH stretching, peak at 2999 cm-1 corresponding to υ(CH) stretching, peak at 2948 cm-1 due to υ(CH2) stretching, peak at 2883 cm-1 due to υ(CH3) stretching, peak at 1759 cm-1 due to C=O stretching of ester, peaks at 1456 and 1385 cm-1 characteristic to asymmetric and symmetric deformation of CH3) and peak at 1187 cm-1 demonstrate C-O-C stretching [51]. For PLA/1S, PLA/1R nanocomposites no significant differences in FTIR spectra of PLA is shown. The only difference in the spectra was observed in peaks characteristic to Ti-O-Ti that was shifted to higher wavenumber, confirming the physical interaction between TiO2 nanoparticles and PLA. The peaks characteristic to Norfloxacin at 3445 cm-1 (–OH stretching), 2918 cm-1 (=CH and aromatic C-H stretching), 2838 cm-1 (CH2 stretching), 1735 cm-1 (C=O stretching of carboxylic group, 1625 cm-1 (N-H bending of quinolones), 1394 cm-1, 1339, 1267 cm-1 (O-H bending), 1092, 1031 cm-1 (C-F bending), 931 cm-1 (N-H bending of amine) , 826 cm1

(distribution of aromatic protons) are shown in Fig. 2 [52]. In NOR@TiO2 (S),

characteristic peak of TiO2 at 668 cm-1 was shifted to 693 cm-1. Norfloxacin can form a coordination complex with the surface of TiO2 [52]. FTIR spectrum of NOR@ 1S/En PLA shows slight shift in peak characteristic to TiO2 due to physical interaction between TiO2, and NOR. Absence of new bands at 1554 and 1421 cm-1 in 15

nanocomposites confirm the absence of bidentate coordination bond between Ti atoms and the carboxylic groups of PLA. The SEM micrographs of TiO2 (S, R) nanoparticles prepared by different routes are shown in Fig. 3. SEM image of TiO2 (S) nanoparticles (Fig. 3a) prepared via sol gel method shows spherical shape. It was reported that [36] hydrolysis of Titanium Tetra Isopropoxide produced spherical nanoparticles due to acidic condition that prevent agglomeration. The morphology and size of TiO2 particles is affected by the water to titanium molar ratio. It was reported that spheres with diameter range from 0.5 to 1 mm when the molar ratio ≤ 10 was obtained. However, nanoparticles less than 100 nm in diameter were formed under specific condition inducing higher molar ratio and pH level of the solution not exceed 2 [53]. The as prepared powder by hydrothermal method (Fig.3b) consists of rods with an average diameter of 70 nm and length of 400 nm, approximately 3-4 crystallites. Rasin et al. [38] was reported that nanorods and nanotubes were partially formed at 100oC while converted totally at 1D structure above 150oC with average diameter not far from the starting nanoparticle diameter. TEM images of TiO2 (S, R) (Fig. 3c, d) confirm the spherical and rod morphology with an average diameter around 27 nm and 30 nm respectively. SEM image of fracture surface of pure PLA (Fig. 4a) shows brittle fracture pattern. It is worth noting that the SEM image of a cross-sector of PLA/3S (Fig. 4b) shows small white spots appear in the image of the fracture surface indicating that TiO2 dispersed well in the matrix using sonication without any modification. The detailed dispersion of TiO2 nanoparticles in the PLA matrix were determined by TEM. The nanospheres and nanorods have been uniformly distributed in the PLA/3S (Fig.4c) and PLA/3R (Fig.4d) respectively. Sonication can break the intermolecular interaction leading to break up aggregates of micron-sized particles [54]. Recently, the same morphology 16

was observed in PLA/TiO2 prepared by melt blending with a vane extruder [55]. Two types of agglomeration, soft and hard, should be overcome during the preparation process. Soft agglomeration caused by electrostatic and Vander Waals force which are weak and can be overcome by mechanical process. However, hard agglomeration is caused by stronger chemical bonds which are not easy to overcome. In this work, we used ultrasonication dispersion which eliminate these kinds of agglomeration [31]. TEM images of NOR@ 3S/En PLA (Fig.4e, f), clearly show the TiO2 (S) embedded inside the PLA vesicles due to the appearance of dark spheres inside the vesicles and some incorporated nanospheres are attracted on the surface of vesicles. The influence of TiO2 (S) nanoparticles on the degree of crystallinity and thermal stability of PLA was demonstrated by DSC and TGA analysis. Fig. 5A shows the DSC for PLA, PLA/1S and PLA/5S. The addition of TiO2 did not result in a remarkable change in Tg (60 ⁰C) due to relatively low contents of TiO2. The melting temperature of PLA (170 oC), melting enthalpy (38.7 J/g) (Table 1) were influenced in PLA/5S, to be 172 ⁰C, 45.13 J/g, respectively. Also, the crystallization percentage was improved by 10% without a visible peak due to the nucleation effect of TiO2 [33]. Fig. 5B shows the thermogravimetric curves of PLA and PLA/ TiO2 nanocomposites. Pure PLA exhibited a peak decomposition temperature (Td) at 298oC; it was clearly shown that the onset degradation temperature of nanocomposites (303, 308 and 314 oC) was improved compared to pure PLA due to the barrier properties of TiO2 in the early stages of thermal decomposition. Improvement of thermal stability with increasing the TiO2 concentration was referred to the fact that TiO2 absorbs heat energy and retards PLA degradation by blocking the volatile degradation products. Also, reduce the mobility of PLA 17

molecular chain. In addition, high concentration of TiO2 may act as insulator and may transport barrier to the volatile products that produced during decomposition [56]. The amount of char at 500oC increases by increasing the feeding amount of TiO2. 3.2.

Loading of NOR onto carriers

The drug loading efficiency was found to be 30.75±0.5% in PLA after 6h and slightly improved to be 30.58±0.58%, 31.73±0.73%, and 37.5±0.5% in PLA/1S, PLA/3S and PLA/5S respectively. Interestingly the loading was improved to be 35.88±0.2%, 37.6±0.6%, and 40.77±0.78% in PLA/1R, PLA/3R and PLA/5R respectively as shown in Fig. 6. Generally the loading % increased by increasing the percentage of TiO2 in the nanocomposites. The nanocomposites contain TiO2 (R) have higher loading (%) than nanocomposites contain TiO2 (S) due to large surface area of TiO2 (R) nanoparticles which permit more drug molecules to be adsorbed on its surface. Claus Moseke et al [57] reported that higher amount of antimicrobial drugs could be adsorbed on Ti surfaces modified with TiO2 nanotubes in comparison with nonmodified Ti surface. The capacity of TiO2 (S) nanoparticles as a drug carrier for loading NOR drug was studied. Different parameters were studied on loading efficiency of NOR@TiO2 nanoparticles. Fig. 7 shows the effect of Time, Amount of adsorbent, pH of medium and temperature on loading NOR onto TiO2 (S) nanoparticles. To study the effect of adsorbent amount 0.01, 0.03, 0.05 g of TiO2(S) were tested using 14 ppm of NOR, at pH= 6.8±0.2, 30 ⁰C, the loading % increased with time and reached 94.33±0.5% for 0.05g TiO2 after 85 min (Fig. 7A). However, for 0.03g, 0.01g the capacity reached 90.96%±1.84, 84.02±1.73% after 130 min, 300 min respectively. By increasing amount of TiO2 (S) nanoparticles, loading efficiency was increased, this is due to increase numbers of binding sites for adsorption and 18

saturation occurs as a result of non-availability of drug molecules. To study the effect of drug concentration (10-18 ppm), 0.01g of TiO2 at pH 6.8, 30 ⁰C was tested. By increasing concentration of drug, loading efficiency decrease, this is due to deficiency of active sites on carrier to be available for more drug molecules at higher concentrations. To study the effect of pH (2,7,10), 14 ppm of NOR, 0.01 g of TiO2 at 30⁰C were tested. The adsorption of NOR molecules is high at pH= 7 and decreased at pH=2, 10. At pH=2 protonation occur to drug molecules which decrease affinity of molecules to interact with nanoparticles, so, loading efficiency decrease. At pH=10, OH- ions compete with drug molecules for binding to TiO2 nanoparticles, so, loading efficiency decrease. It is worthy to note that Norfloxacin drug is highly soluble in pH˂4 and pH˃8, It has two pKa, pKa1 (6.2-6.4), pKa2 (8.7-8.9). The adsorption rate of NOR onto TiO2 is very low at low pH< 6.2, the zwitter ionic structure of NOR was between 6.2 and 8.0 and anionic form exist at pH higher than 6.5. The adsorption rate starts to increase at pH higher than 4 onto anatase TiO2 due to increasing the Zwitter ionic form of NOR [52]. To study the effect of temperature (25-65⁰ C), 4ppm of NOR, 0.01g of TiO2, pH=6.8 was tested. By increasing temperature, the tendency of drug molecules to interact with nanoparticles decrease, so, loading efficiency decrease, confirming that adsorption process is an exothermic process. Adsorption isotherm models were applied onto NOR loaded TiO2 (S), R2 was calculated to be 0.993 and 0.999 for Langmuir and Freundlich isotherm models individually confirming that adsorption process follow Freundlich isotherm model. Also, Adsorption kinetic models were applied onto NOR loaded TiO2 (S), R2 was calculated to be 0.758, 0.986 for first and second order respectively confirming that adsorption of drug onto TiO2 (S) nanoparticles follow Pseudo-second order kinetic model (Table S1). 19

Release of NOR The in vitro release behavior of NOR from NOR@PLA, NOR@PLA/5S, NOR@PLA/5R and NOR@5S/En PLA carriers was carried out in pH=6.7, 7.4 to simulate the pH of small intestine fluid and physiological blood respectively. A roughly constant release of NOR from PLA/5S, PLA/5R and 5S En PLA nanocarriers is shown in Fig. 8. At pH 7.4 (Fig.8 A) the release continued to develop 70% from PLA, 52% from PLA/5S, 80% from PLA/5R within 240h. However, in NOR @ 5S/En PLA the release continued up to 1080 h to release 90%. The potential for prolonged NOR retention time in blood circulation, reduce the side effects to normal cells effectively [58]. The poor release rate in the encapsulated form was related to the crystallinity of PLA membrane which limits the diffusion of the aqueous buffer environment into the polymer inner layers and limits the diffusion of the drug from the film. The mechanism of drug release was studied by fitting the initial 60% of the drug released from NOR @ 5S/En PLA with Korsmeyer Peppas model. The n value is lower than 0.45 confirm that the release behavior followed Fickian diffusion mechanism (Table S2). It is obvious that release of drug from carriers in slightly acidic medium is faster than release in neutral medium as shown in Fig. 8 B. This was related to presence of H+ ions in acidic medium that compete with active site in carriers for binding with lone pair of electrons of (N) atom present in NOR drug and make protonation for it. Thus H-bond formation between drug and carriers decreased, so, drug released faster to the buffer solution. This behavior facilitate the accumulation of drug in acidic extracellular tumor environment and intracellular endosomal and lysosomal compartment leading to improvement the anticancer activity in comparison with conventional drug due to the high concentration of the drug in the site of infection causing the cell death [59]. It is worth noting that the 20

release behavior of NOR from NOR@TiO2 was rapid in different pHs media (Fig. S1). Within 48 h more than 40% of NOR was released. Daunorubicin (DNR) loaded TiO2 nanoparticles exhibited fast release in acidic pH and reached 86% after 48h due to the repulsion force between positively charged drug and TiO2 nanoparticles that accepted positive charge at lower pH [59]. Adriamycin (ADR) containing poly(Llactic acid) microsphere showed roughly constant release of ADR from microspheres continued to 20h to release 100% [60]. It was found that the most release mechanism followed Higuchi kinetic model (Table S2, Fig.S2) in different pH media. 3.3.

Antibacterial activity study

Carriers loaded and encapsulated NOR drug was tested for antibacterial activity against Gram +ve bacteria such as: Staphylococcus aureus and Gram -ve bacteria such as: Pseudomonas aeruginosa, Salmonella, Escherichia coli and Klebsiella pneumonia using Norfloxacin drug as a standard. The results of the antibacterial efficacy tests are shown in Fig. 9. It was observed that PLA loaded NOR has antibacterial activity against Gram + ve and Gram –ve bacteria. PLA/TiO2 nanocomposites containing TiO2 (R) show more efficacy in killing bacteria than PLA/TiO2 nanocomposites containing TiO2 (S), This may be attributed to high loading % and faster release behavior. In addition, by increasing TiO2 concentration in PLA matrix, nanocomposites show better behavior in killing bacteria. The available TiO2 surface area and homogenous dispersion of TiO2 (S, R) within PLA matrix are the key features in optimizing bio killing activity in PLA/TiO2 nanocomposites [61]. NOR loaded carriers have ability to sustain their antibacterial activity as same as NOR but better than Norfloxacin drug in some species as Salmonella and E.coli [62]. The efficacy of encapsulated carriers is lower than adsorbed carriers due to the delay in the release of drug within 24h as shown in Fig. 8. Porous PLA/TiO2 nanocomposite 21

films showed improved antibacterial activity against S. aureus and used for wound dressing application [63]. It was reported that higher availability of TiO2 nanoparticles at the surface of polymer enhances the bactericidal activity. The bactericidal property depends on the size, surface area and morphology. Nano size with high surface area imparts good bactericidal property [63]. Many studies have shown that reactive oxygen species that are produced by aerobic cells during metabolism can destroy cell membrane constituents of microorganism and damage the integrity of membrane. The electrostatic force between positively charged TiO2 nanoparticles and bacterial surface can promote lipid peroxidation of the cell membrane; increasing cell membrane permeability and once the membrane are damaged, TiO2 NPs can enter the cell and bind to biological macromolecules resulting in protein denaturation [64]. Many studies on the toxicity of TiO2 for microorganisms found that the toxicity varied with particle size and specific to species [64]. It was observed that TiO2 can travel in the blood stream by binding to plasma protein, through the lymphatic system after phagocytosis by macrophages or to the bone marrow via monocytes [65]. 3.4.

Cytotoxicity assay

The cytotoxicity efficacy of carriers incapsulated NOR is shown in Fig.10. The x-axis shows cell types with varying carriers (drug delivery systems), while the y–axis shows inhibition rates of cancer cells. NOR@ 5S/En PLA was found the most active carrier against different cell lines compared with NOR against 5 tumor cell lines HePG-2, HCT-116, MCF-7, PC-3 and Hela. It was reported that TiO2 nanoparticles have a high effect on breast cancer cell lines [66]. The cytotoxicity efficacy was related to ROS production that produced using electron capture pathway [67]. These nanoparticles play an important role in DNA damage, membrane devastation and finally cell death via oxidative stress and lipid peroxidation [68]. Many studies have 22

shown aggregated crystals of TiO2 residue in mitochondria causing defect in electron chain and destruction its function. The degree of toxicity of TiO2 nanoparticles depend on the concentration and time of exposure [69]. TiO2 can bail up via binding to plasma proteins that contain OH, NH and NH2 groups in the blood stream to the bone marrow [70]. Interestingly, in normal cell lines as WISH and WI-38, DOX destroyed normal cells of human body very strongly but these biocarriers has moderate cytotoxic effect on normal cells. In WI-38 normal cell, NOR@5S/En PLA has lower cytotoxic effect in comparison to NOR itself and this is good in delivery of cancer drugs. TiO2 nanoparticles loaded chemotherapeutic agents showed anticancer efficiency enhancement [19,68,74] due to accumulation, leaving blood vessels through small pores present in vessels, of drug in the tumor cells. The in vivo study and photocatalytic activity upon irradiation on these nanocomposites are under investigation. 4.

Conclusions

TiO2 with different morphologies were successfully synthesized via sol gel and hydrothermal method. The crystallites are aggregated to form spheres and rod like structure. PLA/TiO2 nanocomposites were prepared by sonication process followed by casting film. NOR drug was loaded onto TiO2 nanospheres followed by encapsulation into PLA. In addition, the loading capacity of NOR onto PLA/TiO2 nanocomposites using different contents of TiO2 (1, 3, 5 wt %) and different morphologies (spheres, rods) were studied and compared with loading onto PLA. No considerable change in the IR peaks of NOR, PLA indicating the absence of chemical interaction between drug and carriers. Results indicated that the high loading efficiency of NOR onto TiO2 was done under pH= 7, amount of TiO2 nanospheres =0.05 g, NOR concentration=10 ppm and contact time=300 min. Pseudo-second order 23

model was more adequate to represent the kinetic data and the equilibrium data followed Freundlich model. It was observed that encapsulation of drug delays release time than adsorption of drug on the surface of nanocomposites. Antibacterial activity of drug loaded by adsorption process have higher efficacy than encapsulation process of drug. However, encapsulated drug showed the highest efficacy compared with NOR against 5 tumor cell lines with moderate cytotoxic effect on normal cells. The results justify the formulation of NOR@5S/En PLA is a good alternative and suitable carrier for tumor cell lines with moderate cytotoxicity efficacy on normal cell line and the formulation exhibit desired antibacterial activity for treatment bacterial infections. Conflict of Interest The authors declare no conflict of interest. ORCID Nehal Salahuddin: 0000-0002-8501-3681 Mohamed Abdelwahab: 0000-0002-8094-3644

Figures Caption Fig. 1: X-ray diffraction patterns of (A) TiO2 (S), and TiO2 (R), (B) PLA, PLA with 1, 2 and 3%S, (C) PLA and PLA with 1, 3 and 5%R and D) NOR@TiO2 (S) and NOR@S/En PLA nanocomposites. Fig. 2: FTIR spectra of a) TiO2 (S); b) TiO2 (R); c), PLA; d) PLA/1S; e) PLA/1R; f) NOR; g) NOR@ TiO2 (S); h) NOR@ 1S/En PLA. Fig. 3: Scanning electron micrographs of TiO2 (S) (a), TiO2 (R) (b), Transmission electron micrographs of TiO2 (S) (c), TiO2 (R) (d).

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Fig. 4: Scanning electron micrographs of PLA (a), PLA/3S (b), Transmission electron micrographs of PLA/3S (c), PLA/3R (d), NOR@ 3S/En PLA (e, f) at different magnifications. Fig. 5: Differential scanning calorimetry of (A) PLA, PLA/3S, PLA/5S (d) and Thermal gravimetric analysis of (B) PLA (a), PLA/1S (b), PLA/3S (c), PLA/5S (d). Fig. 6: NOR Loading (%) onto PLA and PLA/TiO2 (S, R) using different contents and morphology of TiO2. Fig. 7: The effect of NOR concentration, pH of medium, Temperature and amount of TiO2 on loading % of NOR onto TiO2 (S). Fig. 8: In vitro release behavior of NOR from PLA, PLA/5S, PLA/5R and NOR@5S/EnPLA at different pH media A) pH= 7.4; B)6.7. Fig. 9:

Inhibition zones (mm) of a. NOR (control), b. NOR@PLA, c.

NOR@PLA/1S, d. NOR@PLA/3S, e. NOR@PLA/5S, f. NOR@PLA/1R, g. NOR@PLA/3R, h. NOR@PLA/5R, i. NOR@ 1S/En PLA, j. NOR@ 3S/En PLA, k. NOR@ 5S/En PLA against different bacterial strains. Fig. 10: In vitro cytotoxicity of a) Doxorubicin (control), b) Norfloxacin, c) NOR@TiO2 (S), d) NOR@1S/En PLA, e) NOR@3S/En PLA, f) NOR@5S/En PLA against different cancer cell lines and normal cell lines. Where, IC50 (µg/ml): 1-10 (very strong), 11-20 (strong), 21-50 (moderate), 51-100 (weak) and above 100 (non cytotoxic).

Supporting information: Fig.S1: Release behavior of NOR from NOR@TiO2 (S) at different pH media

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Fig.S2: First order (a); Higuchi (b); Hixon-Crowell (c) and Krosmeyer-Peppas linear fit of NOR release from A) PLA, B) PLA/5S, C) PLA/5R, D) NOR@5S/En PLA. Table 1: Thermal properties (TGA and DSC data) of PLA and PLA with different ratio of TiO2. Table S1: Adsorption kinetic parameters and adsorption isotherm parameters for adsorption NOR onto TiO2 (S). Table S 2: Controlled release kinetics of NOR from PLA, PLA/5S, PLA/5R adsorbed NOR and NOR@5S/En PLA at different pH media.

5.

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Synthesis and Design of Norfloxacin Drug Delivery System Based on PLA/TiO2 Nanocomposites: Antibacterial and Antitumor Activities Nehal Salahuddin*, Mohamed Abdelwahab, Mohamed Gaber, Sahar Elneanaey Chemistry Department, Faculty of Science, Tanta University, Tanta, 31527 Egypt

Figures

Fig. 1: X-ray diffraction patterns of (A) TiO2 (S), and TiO2 (R), (B) PLA, PLA with 1, 2 and 3%S, (C) PLA and PLA with 1, 3 and 5%R and (D) NOR@TiO2 (S), PLA and NOR@S/En PLA nanocomposites.

Fig. 2: FTIR spectra of a) TiO2 (S); b) TiO2 (R); c), PLA; d) PLA/1S; e) PLA/1R; f) NOR; g) NOR@ TiO2 (S); h) NOR@ 1S/En PLA.

Fig. 3: Scanning electron micrographs of: (a) TiO2 (S); (b) TiO2 (R); Transmission electron micrographs of (c) TiO2 (S); and (d) TiO2 (R).

Fig. 4: Scanning electron micrographs of (a) PLA; (b) PLA/3S, Transmission electron micrographs of (c) PLA/3S; (d) PLA/3R; (e, f) NOR@ 3S/En PLA at two different magnifications.

Fig. 5: Differential scanning calorimetry of (A) PLA, PLA/3S, PLA/5S; and (B) Thermogravimetric analysis of: (a) PLA; (b) PLA/1S; (c) PLA/3S; (d) PLA/5S.

Fig. 6: NOR Loading (%) onto PLA and PLA/TiO2 (S, R) using different contents and morphology of TiO2.

Fig. 7: The effect of NOR concentration, pH of medium, Temperature and amount of TiO2 on loading % of NOR onto TiO2 (S).

Fig. 8: In vitro release behavior of NOR from PLA, PLA/5S, PLA/5R and NOR @5S/En PLA nanocomposites using different pH media: a) pH= 6.7, b) 7.4.

Fig. 9: Inhibition zones (mm) of a. NOR (control), b. NOR@PLA, c. NOR@PLA/1S, d. NOR@PLA/3S, e. NOR@PLA/5S, f. NOR@PLA/1R, g. NOR@PLA/3R, h. NOR@PLA/5R, i. NOR@ 1S/En PLA, j. NOR@ 3S/En PLA, k. NOR@ 5S/En PLA against different bacterial strains.

Fig. 10: In vitro cytotoxicity of a) Doxorubicin (control), b) Norfloxacin, c) NOR@TiO2 (S), d) NOR@1S/En PLA, e) NOR@3S/En PLA, f) NOR@5S/En PLA against different cancer cell lines and normal cell lines. Where, IC50 (µg/ml): 1-10 (very strong), 11-20 (strong), 21-50 (moderate), 51-100 (weak) and above 100 (non cytotoxic).

Table 1: Thermal properties (TGA and DSC data) of PLA and PLA with different ratio of TiO2. Code

Td (oC)

Tm (oC)

∆H (J/g)

Xc (%)

PLA

298

170

38.70

41.56

PLA/1S

303

172

35.00

37.97

PLA/3S

308

170

33.13

36.68

PLA/5S

314

172

45.13

51.03

Td is the onset decomposition temperature; Tm is the melting temperature, ∆Hm is the enthalpy of melting, and Xc is the degree of crystallinity of PLA.

Highlights 1. PLA/TiO2 nanocomposites were prepared using TiO2 nanospheres and nanorods by sonication technique. 2. The optimum loading of NOR onto TiO2 nanospheres was studied. 3. The adsorption of NOR onto TiO2 nanospheres fits well with pseudo-second order model and freundlish isotherm. 4.

The release of NOR from TiO2 loaded encapsulated into PLA and NOR adsorbed onto nanocomposites was compared.

5. PLA/TiO2 nanorods formulations exhibited high antibacterial efficacy, However, NOR loaded TiO2 nanospheres encapsulated PLA formulation exert high antitumor efficacy.

Synthesis and Design of Norfloxacin Drug Delivery System Based on PLA/TiO2 Nanocomposites: Antibacterial and Antitumor Activities Nehal Salahuddin*, Mohamed Abdelwahab, Mohamed Gaber, Sahar Elneanaey Chemistry Department, Faculty of Science, Tanta University, Tanta, 31527 Egypt

Conflict of Interest The authors declare no conflict of interest.