Iraqi J Pharm Sci, Vol.29(2) 2020 5Fluorouracil nanosponges DOI: https://doi.org/10.31351/vol29iss2pp88-98 88 Synthesis and Evaluation of β-cyclodextrin Based Nanosponges of 5- Fluorouracil Using Ultrasound Assisted Method. Ihsan K. Jasim,1, Shaimaa N. Abd Alhammidand Alaa A. Abdulrasool   Ministry of Health and Environment, Babylon Health Directorate, , Babylon, Iraq. Department of Pharmaceutics, College of Pharmacy, University of Baghdad, Baghdad, Iraq. Abstract 5-Fluorouracil (5-FU) is mostly used in the treatment of stomach cancer. After intravenous injection of 5-FU, it is rapidly distributed and eliminated with an apparent terminal half-life of 8-20 min. It is poorly absorbed after oral administration with an extremely variable bioavailability. Hence, this study has been made to synthesize 5-FU nanosponges (NS) to increase its accumulation in gastric tumors by the help of an enhanced permeability retention effect (EPR) and decrease its systemic side effects. On the other hand, 5-FU is sparingly soluble in water so its dissolution can be increased by incorporation in nanosponges as nanoparticles. CD-nanosponges were prepared by crosslinking β-CD with diphenylcarbonate (DPC) using ultrasound assisted technique. 5-FU was incorporated with NS by freeze drying, and the phase solubility study, complexation efficiency (CE) entrapment efficiency were performed. Also, the particle morphology was studied using SEM and AFM. The in-vitro release of 5-FU from the prepared nanosponges was carried out in 0.1N HCl. 5-FU nanosponges particle size was in the nano size. The optimum formula showed a particle size of (405.46±30) nm, with a polydispersity index (PDI) (0.328±0.002) and a negative zeta potential (-18.75±1.8). Also the drug entrapment efficiency varied with the CD: DPC molar ratio from 15.6 % to 30%. The SEM and AFM showed crystalline and porous nature of the nanosponges. The in vitro drug release study of the selected formula 5-FUNS2 exhibited the fastest dissolution rate which is 56% in the first hr. Different molar ratios of (cyclodextrin to crosslinker) (CD: DPC) has a proficient effect on complexation efficiency (CE), apparent stability constant (Kst) and entrapment efficiency of 5-FU. 5-FUNS2 with (1:4) molar ratio showed the best result of CE, Kst and entrapment efficiency. 5- FUNS2 gave a higher release rate than the 5-FU-βCD inclusion complex and 5-FU solution. Surface morphology of the prepared nanosponges by SEM, AFM indicate that nanosized and highly porous nanosponges was obtained. The overall results suggest that cyclodextrin nanosponges could be a promising 5-FU delivery system utilizing the suitable formula. Keywords: 5-FU, nanosponges, ultrasound assisted method, βCD, DPC, SEM, AFM. البيتا سايكلودكستيرين باستخدام فلورويوراسيل-5 جسيمات اإلسفنجية النانوية لعقار وتقييم تحضير الصوتية فوق بمساعدة الموجات **عالء عبد الحسين عبد الرسول و ** شيماء نزار عبد الحميد ،1*،احسان خضير جاسم بابل ،العراق. ،دائرة صحة بابل ،والبيئة وزارة الصحة * بغداد،العراق. جامعة بغداد، كلية الصيدلة، فرع الصيدالنيات ،** الخالصه مع تخلص منه يتم الفي الوريد ، يتم توزيعه بسرعة و حقنهالمعدة . بعد يستخدم بشكل شائع في عالج سرطان FU-5فلورويوراسيل 5 متغير للغاية. وبالتالي ، في حيويبعد تناوله عن طريق الفم مع توفر ضعيففلورويوراسيل بشكل 5دقيقة. يتم امتصاص 20-8 عمر نصف تبلغ نفاذيةبالحتفاظ عن طريق المساعدة في تعزيز تأثير اال أورام المعدةلزيادة تراكم الدواء في FU-5 من إسفنجيةهذه الدراسة قد تم تصنيع جسيمات (EPR) عن هفلورويوراسيل شحيح الذوبان في الماء. يمكن زيادة معدل ذوبان 5. أيضا ، الناتجة عن انتشار الدواء في الجسم وتقليل اآلثار الجانبية .سيكلودكستيرين البيتا-النانوإسفنجيات في هطريق تضمين عن طريق ربط باواصر تساهمية للبيتا سايكلودكسترين مع الكربونات ثنائية وية جسيمات البيتا سيكلودكستيرين اإلسفنجية النانتم تصنيع داخل الجسيمات اإلسفنجية النانوية بواسطة طريقة التجفيف بالتجميد وكذلك تم إنجاز دراسة FU-5 تم دمجالموجات فوق الصوتية. الفينول بمساعدة جهد زيتا باستخدام محلل زيتا بلس. تمت دراسة و الجزيئات حجومتم قياس . كما الدواءحجز كفاءة و (CE) كفاءة تكوين المعقدطور الذوبانية و النانوية في محلول حامض اإلسفنجيةمن الجسيمات FU-5تحرر الدواء داخل المختبر لـ قياستم و AFMو SEMشكل الجسيمات أيًضا باستخدام .0.1 عياريبتركيزالمخفف ك يالهيدروكلور 1Corresponding author E-mail: ihsan.aljanaby@yahoo.com Received: 10/1 /2020 Accepted: 3/ 5/2020 Iraqi Journal of Pharmaceutical Science https://doi.org/10.31351/vol29iss2pp88-98 Iraqi J Pharm Sci, Vol.29(2) 2020 5Fluorouracil nanosponges 89 ( نانومتر 30± 405.46كان في حجم النانو ، الصيغة المثلى توضح حجم الجسيمات ) FU-5النانوية لعقار اإلسفنجية حجم الجسيمات PDI(,(0.328 ± 0.002 ( (. تباينت معدل الدواء المحتجز مع النسبة المولية )1.8± 18.75-وجهد زيتا سالبةCD: DPC من )2.6± 15.6 ٪ في للصيغة المثلى . أظهرت دراسة التحرر الدوائي اإلسفنجيةالطبيعة البلورية والمثقبة للجسيمات AFMو SEM٪. وأظهرت 2.3± 30إلى ٪ في الساعة األولى. 56 تحررمعدل أسرع المختبر و Kstو CE( لها تأثير كبير على معدل CD: DPC( )المكون للروابط التساهميةالى دكستيرين البيتا سيكلوالنسب المولية المختلفة لـ ) FUNS2-5حتجاز الدواء. يوفر ونسبة ا CE ،Kst( يظهر أفضل نتيجة ل 4: 1)ة نسبة المولي ذو FUNS2-5كمية الدواء المحتجزة .الصيغة الحجم النانو توضح و SEM ،AFMالنانوية المحضرة بواسطة لإلسفنجةالتشكل السطحي . FU-5ومحلول(FU-βCD-5)معدل تحرير أعلى من . ونسبة الثقوب العالية .تسليم الدواء باستخدام الصيغة المناسبةلكون نظام واعد تيمكن أن لبيتا سيكلودكستيرين النتائج اإلجمالية تشير إلى أن الجسيمات النانو ل .βCD ،DPC ،SEM ،AFM، طريقة الموجات فوق الصوتية المساعدة ، اإلسفنجية، الجسيمات النانوية فلورويوراسيل 5 الكلمات المفتاحية : Introduction Although chemotherapeutic agents can reduce tumor size and cancer remission and have a high potential to destroy cancer cells, they are not organ specific and can damage proliferative cells(1). One of the major goals of cancer therapeutics is to kill cancer cells without damaging normal tissues. One way to achieve this is the use of molecularly targeted therapy combined with chemotherapy. Tissue and cell distribution of cancer therapeutic drugs can be controlled by the entrapment in sub-micron level (˂1 µm) colloidal systems, in other words known as nanoparticles. Some of the desirable characteristics that are needed to deliver therapeutic agents to tumor cells include the ability to overcome drug resistance at the tumor and cellular levels and ensure an appropriate distribution, biotransformation, and clearance of the drug (2). Conventional chemotherapeutic agents work by destroying rapid dividing cells, which is the main property of neoplastic cells. This is why chemotherapy also damages normal healthy cells that divide rapidly such as cells in the bone marrow, macrophages, digestive tract, and hair follicles (3). Nanosponges are hyper-cross-linked cyclodextrins that can be obtained with α, β and γ cyclodextrins, either alone or as mixtures containing relevant amounts of linear dextrin, cross-linked with a suitable cross-linking molar ratio, by using an active carbonyl compound, e.g., diphenyl carbonate, by ultrasound-assisted synthesis. Thus, spherical nanosponges of submicron size of cyclodextrin are connected by nanochannels to form a cage-like structure. These nanosponges can be inclusion complex drug carriers(4). Nanotechnology have been applied to improve drug delivery and to overcome some of the problems of drug delivery for cancer treatment (5). Nanosponges are a novel class of hyper-cross linked polymer based colloidal structures consisting of solid nanoparticles with colloidal and nanosized cavities. Nanosponges solubilizes poorly water soluble drugs and provides a prolong release as well as improves the drug bioavailability by modifying the pharmacokinetic parameters of active constituents (6, 7). 5-Fluorouracil (5-FU) was most commonly used in the treatment of cancers of colon, breast, stomach and pancreas (8). However, like other drugs used for chemotherapy, it affects the growth of normal body cells and often causes side effects such as hair loss, fatigue, birth defects, mouth sores, liver disease, and a temporary drop in bone marrow function (9). After intravenous injection of 5-FU, it is rapidly distributed and eliminated with an apparent terminal half-life of 8-20 min with a pKa of 8, and 13 , LogP(-1) (10). Also 5-FU is poorly absorbed after oral administration with an extremely variable bioavailability (11). The aim of the study was synthesis and evaluation of 5-FU loaded nanosponges to enhance the dissolution rate of the sparingly soluble 5-FU. Also nanosponge increases its accumulation in gastric tumors by the help of an enhanced permeability retention effect (EPR) and decrease its systemic side effects. As it is intended to be formulated as floating tablet for local gastric cancer therapy in the future study. Materials and Methods Materials 5-Fluorouracil, βCD and diphenyl carbonate (DPC) were obtained from Hyper-chem Ltd Co. (Hangzhou, China). All other analytical reagents were of analytical grade. Methods Preparation of β-CD-nanosponges using ultrasound assisted method Accurate amounts of βCD and diphenyl carbonate DPC were mixed in 100ml beaker at a different molar ratio as shown in Table (1). The beaker was then placed in an oil bath and heated to 90ᵒC.Then the mixture was sonicated for 4 hours at 50% amplitude using ultrasound probe capable of supplying maximum power of 500 Watt at 20 kHz (Qsonica, USA). The reaction mixture is left to cool and the product obtained is broken up roughly. Numerous needle-shaped crystals of phenols can be seen on the clear surface of the beaker as shown in Figure (1) and part of the phenol developed contributes to agglomerating of the product (12). Iraqi J Pharm Sci, Vol.29(2) 2020 5Fluorouracil nanosponges 90 Subsequently after cooling, the product was broken up roughly by mortar and repeatedly washed with an excess amount of distilled water (DW) through filtration by the Buchner funnel to remove unreacted βCD. An additional purification step consists of Soxhlet extraction in ethanol, which was performed for 24 hours to remove the unreacted DPC and phenol present as by-product of the reaction. Finally, the nanosponges (NS) were dried at room temperature to obtain a fine white powder (13-16). Figure1. a) Structural representation of reaction for preparation of NS, b) Ultrasound assisted method Table1. The composition and molar ratios of CD: DPC used to prepare βCD-NS*. * β-CD β-cyclodextrin, DPC Dipenyl carbonate.**1 mole of β-CD =1.135 g, 1mole of DPC = 0.2142g.***NS1-NS5 plain NS Preparation of 5-FU loaded nanosponges 5-Fluorouracil loaded NS were prepared by freeze drying method(17). Briefly, the prepared βCD nanosponges at different CD:DPC molar ratios and an excess amount of 5-FU as a powder were mixed and the resultant mixture was suspended in 30 ml distilled water. Then, the mixture was sonicated for 10 min and stirred for 24 hours using a magnetic stirrer (Copley, Germany). The obtained aqueous suspension was centrifuged for 10 min at 2000 rpm to separate the uncomplexed drug as a deposit. The supernatant was then lyophilized by employing a lyophilizer (Copley, Germany) to get 5-FU loaded (18). Preparation of 5-FU inclusion complexes One formula of β-cyclodextrin inclusion complex with 5-FU (5-FU–βCD) was prepared. A weighted amount of 5-FU was finely suspended in a water solution containing an equimolar amount of βCD and 5-FU (1:1). The aqueous suspension was then stirred at room temperature in the dark place for 24 h. After centrifugation (5000 rpm, 10 min),(Labent, Germany) then the supernatant was freeze-dried(19). Production yield of the prepared nanosponges Production yield: The production yield can be determined by calculating initial weight of raw materials and final weight of nanosponges obtained (20): 𝐏𝐫𝐨𝐝𝐮𝐜𝐭𝐢𝐨𝐧 𝐲𝐢𝐞𝐥𝐝 = 𝑷𝒓𝒂𝒄𝒕𝒊𝒄𝒂𝒍 𝒎𝒂𝒔𝒔 𝒐𝒇 𝒏𝒂𝒏𝒐𝒔𝒑𝒐𝒏𝒈𝒆𝒔 𝑻𝒉𝒆𝒐𝒓𝒊𝒕𝒊𝒄𝒂𝒍 𝒎𝒂𝒔𝒔 𝒐𝒇 𝒏𝒂𝒏𝒐𝒔𝒑𝒐𝒏𝒈𝒆(𝒑𝒐𝒍𝒚𝒎𝒆𝒓 +𝒄𝒓𝒐𝒔𝒔𝒍𝒊𝒏𝒌𝒆𝒓) × 𝟏𝟎𝟎 Encapsulation efficiency Weighed amount of 5-FU-loaded nanosponges were dispersed in DW and sonicated for 10 min, then centrifuged at 15,000 rpm for 15 min (Copley, Germany) double cycle after that the supernatant was withdrawn, suitably diluted with distilled water and were subjected to UV spectroscopy for measuring the absorbance of the sample at the λmax of 5-FU (266 nm). With the help of absorbance, the concentration in the supernatant was determined by plotting the absorbance value against concentration in the standard curve. Furthermore, the total drug content of 5-FU was determined by dissolving a same amount of 5-FU loaded NS in methanol and sonicated for 10 min to destroy and break the complex to calculate the total amount of 5-FU present in the 5-FU loaded NS powder . The percentage encapsulation efficiency was calculated by following equation(21): % 𝐄𝐧𝐜𝐚𝐩𝐬𝐮𝐥𝐚𝐭𝐢𝐨𝐧 𝐞𝐟𝐟𝐢𝐜𝐢𝐞𝐧𝐜𝐲 = 𝑫𝒓𝒖𝒈𝒆𝒏𝒄𝒂𝒑𝒔𝒖𝒍𝒂𝒕𝒆𝒅 𝑫𝒓𝒖𝒈𝒕𝒐𝒕𝒂𝒍 ∗ 𝟏𝟎𝟎 Phase solubility studies Phase solubility studies were carried out according to the Higuchi–Connors method(22). An accurate amount of 5-FU (100 mg) was added to a series of aqueous solutions (5 mL) containing increasing concentrations of βCD-NS, from (9.5 to 12.7) mM and in βCD (1:1). The samples were stirred in the dark at room temperature for 5 days. After equilibration, the aqueous suspensions were centrifuged and the 5-FU content in the supernatant was determined by UV spectrophotometer at 266 nm. Formula s codes** * β- CD:DPC* * Molar ratio β-CD (g) DPC (g) NS 1 1:2 2.27 0.856 NS 2 1:4 2.27 1.713 NS 3 1:6 2.27 2.57 NS 4 1:8 2.27 3.427 NS 5 1:10 2.27 4.28 Iraqi J Pharm Sci, Vol.29(2) 2020 5Fluorouracil nanosponges 91 The phase solubility diagram was constructed by plotting the total molar concentration of 5-FU against the molar concentration of βCD-NS. Stability constants (Kst) from the phase solubility diagram were calculated using the Equation (1): 𝐊𝐬𝐭 = 𝐬𝐥𝐨𝐩𝐞 𝑺𝟎(𝟏−𝒔𝒍𝒐𝒑𝒆) (1) Where, S0 represents the solubility of 5-FU in the absence of CD. The slope was determined from the initial linear part of the concentration curves of 5- FU. The complexation efficiency (CE) is the concentration ratio between cyclodextrin in a complex and free cyclodextrin, and it was calculated from the phase-solubility diagrams (15). The complexation efficiency is calculated by the slope of the phase-solubility profile using equation 2, which is referred to as the complexation efficiency (CE) (23). CE = S0K1: 1 = D CD⁄ CD = Slope 1 − Slope Since, the numerical value of CE is only dependent on the slope of the phase-solubility profile, less variation is usually observed in the CE values compared to the stability constant Kst value. Characterization of the prepared 5-FU loaded nanosponges Particle size, Polydispersity Index analysis (Dynamic light Scattering ) and zeta potential Nanosponges sizes and polydispersity index were measured by dynamic light scattering using a 90 plus particle sizer (ZetaPlus Particle Sizing, NY, Software, Version 5.34), The samples were suitably diluted with water prior to measurements. Zeta potential measurements were also made using an additional electrode in same instruments. The mean hydrodynamic diameter (Dh) and polydispersity index (PI) of the particles were calculated in intensity using the cumulant analysis after averaging the three measurements (17, 24). Fourier Transform-Infrared Spectroscopy (FT-IR) ATR–FTIR spectra of 5-FU, DPC, βCD, βCDNS and 5-FUNS were recorded on a IRAffinity- S1 Spectrum FT-IR (Shimadzu , Japan) in the region of 4000–650 cm-1. It was performed, using a Shimadzu spectrophotometer, to confirm the formation of βCDNS and understand if there are interaction between drug and NS(15). Scanning Electron Microscopy (SEM) Scanning electron microscopy (Tescan Mira3,France) was significant for determination of surface characteristics and size of the particle. Scanning electron microscope was operated at an acceleration voltage of 15 kV(25). Atomic Force Microscopy (AFM) A further in-depth morphological analysis was performed using an atomic force microscope (Angstrom Advanced Inc. AA3000) with a scanner of 3.1 µm with three piezo electrodes for three axes X, Y and Z in a noncontact mode. The sample suspensions (1% w/v) were prepared in distilled water and a drop was impregnated onto aluminum sheet (2 cm×2 cm). This was allowed to dry in a HEPA filter zone and the dried region was analyzed(26). In-vitro release study of 5-FU nanosponges In-vitro release study of 5-FU from 5-FU- βCD inclusion complex and the selected formula of 5-FUNS was performed by using an accurate amount of impregnated nanosponges equivalent to 100 mg 5-FU suspended in 5 ml of 0.1N HCl solution which was placed in the dialysis membrane (cut off 12,000 Da) and the samples were individually placed in dissolution vessel containing 900 ml of 0.1N HCl, maintained at 37 ± 0.5°C at 75 rpm using a paddle dissolution apparatus (USP Type II). . At various time intervals, aliquots of 5ml were withdrawn and replaced with the same volume of fresh dissolution medium to maintain the sink conditions and the withdrawn samples were analyzed by UV spectrophotometer (EMCLAB, Germany) at 266 nm(27, 28). Statistical analysis The results are reported as the mean±SD and statistical significance was determined using one-way analysis of variance (ANOVA) and Student’s t-tests as appropriate. All experiments were performed in triplicate and values were expressed as the mean standard deviation SD. Values of p < 0.05 were considered statistically significant(29). Results and Discussion Production yield The practical yield of nanosponges was found to be less for lower (β-CD: DPC) molar ratio (1:2). The practical yield increased with the increase in molar ratio up to 1:8, and at higher molar ratios (1:8 - 1:10), the yield was found to be almost the same for both molar ratio. This may be due to saturation of the reactive functional groups at higher concentration(30).the molar ratio significantly (p˂0.05) affected the production yield of CD NS. Iraqi J Pharm Sci, Vol.29(2) 2020 5Fluorouracil nanosponges 92 Table2. Production yield percent of the prepared CD NS , data are expressed as Mean ± SD, n =3, standard deviation (SD). Formulas No.* Theoretical weight (g) Production yield (g)±SD Production yield % NS 1 2.702 1.85±0.19 48.1 NS 2 3.13 3.18±0.3 63.9 NS 3 3.559 4±0.1 70.2 NS 4 3.987 4.72±0.2 72.7 NS 5 4.414 5.64±0.3 74.8 *NS1-NS5= plain nanosponges. Phase solubility study Phase solubility studies were conducted for all the prepared nanosponges and their respective nativeβ-cyclodextrin in DW. The solubility of 5-FU was found to be about (0.02 M) in the absence of β- cyclodextrins nanosponges. Also, the molar concentration of NS was determined by calculated the molecular weight of NS according to the chemical formula that was mentioned in Figure (1). The phase solubility diagram revealed that the solubility of 5-FU increased linearly as a function of increasing cyclodextrin nanosponges concentration indicating the phase solubility profile obtained was an “A-type” diagram, according to the Higuchi and Connors classification(22) . Cyclodextrin-based nanosponges showed superior complexing ability than natural cyclodextrins towards many molecules. The parent βCD complex shows lower complexation efficiency(CE) and apparent stability constant (Kst) (0.22 ) and (10±0.5 M-1) respectively ,as shown in Table (3).These values were lower than that obtained by βCD nanosponges. This gave significant (p˂0.05) differences of CE and Kst values between the prepared CDNS and the parent CD. This is due to the carbonate linkage which was added to the primary hydroxyl groups of the parent βCD unit. Thus, the drug molecules could be included inside the nanocavities of βCD and due to the cross-linking further interactions of the guest molecules with more βCD units might be thought. Moreover, the presence of the cross-linked network might also form nanochannels in the NS structure for the polymer mesh(16). Figures (2a) and (2b) shows the phase solubility diagram of 5-FU with the prepared NS and βCD, respectively. The slopes of the curves of complexes were lower than one, demonstrating the formation of 1:1 inclusion complex. Figure 2.Phase solubility diagram of 5-FU included in ; a) NS, b)βCD Table3. Different parameters of phase-solubility studies of 5-FU with the prepared NS and CD in distilled water, at 25 °C. Formulas codes Phase solubility study* Slope (M- 1) Intercept Sₒ (M) (5-FU solubility) R² Kst (M-1) mean ± SD* Complexation efficiency (CE) 5-FUNS 1 0.3856 0.02 0.936 30.2±1.6 0.63 5-FUNS 2 0.6511 0.02 0.9817 89.5±3.6 1.87 5-FUNS 3 0.5811 0.02 0.8699 66.3±2.5 1.39 5-FUNS 4 0.4502 0.0199 0.9725 41.1±1.4 0.81 5-FUNS 5 0.4358 0.02 0.9702 37.2±3.1 0.77 5-FU-CD Complex 0.18 0.02 0.987 10.7±1.2 0.22 *Data are expressed as mean ± SD, n =3, standard deviation (SD). Iraqi J Pharm Sci, Vol.29(2) 2020 5Fluorouracil nanosponges 93 Encapsulation efficiency Among different types of nanosponges, the encapsulation efficiency of β-CDNS for 5-FU was observed to be higher in 5-FUNS2 ( β-CDNS(1:4) ) as much as 30±2.3% w/w followed by 5-FUNS3( β- CDNS(1:6) ) 25±2% and NS5 ( β-CDNS(1:10) ) 22±2 % as shown in Table 4. The results revealed that the degree of cross-linking affected the encapsulation efficiency of nanosponges with a significant difference (p˂0.05) between 5-FUNS1 and 5-FUNS2. It was found that at 1:2 molar ratio, the degree of cross- linking may be low, resulting in insufficient nanochannels for the guest complexation; thus 5-FU might not be encapsulated in higher amounts. Particle size, polydispersity index analysis and zeta potential The dynamic light scattering (DLS) related measurements were carried out after lyophilization. Table 5 illustrates the particle size values of the prepared 5-FU nanosponges of CD-NS in which the smallest value was (256.3±24nm) and the largest one was (846.83±51nm). The overall sizes of NS found in the submicron range (<1μm) might be due to charge accelerated aggregation and molecular nature of relative CDs, resulting in a size increment. The increased size may be due to the aggregation during the drying process (31). Zeta potential predicts the long term stability of the nanosized formulations(32). Zeta potential as a measure of surface charge was tested for 5-FU nanoformulations that have small particle size and lower PDI (5-FUNS2 and 5-FUNS3). The results of zeta potential obtained are presented in Table 4. Table 4. Characterization of 5-FU nanosponges, (mean±SD) n=3 Formula code CD:DPC Particle size ± SD (nm)* PDI ZP (mV) Encapsulation efficiency 5-FUNS 1 1:2 545.45±23 0.492±0.01 - 15.6±2.6 5-FUNS 2 1:4 405.46±30 0.328±0.002 -18.75±1.8 30±2.3 5-FUNS 3 1:6 435.43±18 0.464±0.02 -16.1±1.2 25±2 5-FUNS 4 1:8 846.83±51 0.359±0.01 - 19±1.2 5-FUNS 5 1:10 256.3±24 1.711±0.1 - 22±1.7 On the basis of particles size, polydispersity index, zeta potential and encapsulation efficiency formulas 5-FUNS2 was chosen as the optimized formula for the preparation of nanosponges. Drug- excipients compatibility studies The spectrum of 5-FU shows characteristic absorption bands in the region between 1656 and 1723 cm-1 correlated to the C=C, C=N, C=O, while the region at1247–1425 cm-1 was assigned at the vibration of the substituted pyrimidine. The bands at 470, 551, 642, 749, and 813 cm-1, as well as those between 2407 and 3100 cm-1 are due to the aromatic ring(33), as shown in Figure(3). Figure 3. FTIR of 5-FU Iraqi J Pharm Sci, Vol.29(2) 2020 5Fluorouracil nanosponges 94 The appearance of the new peak of the carbonyl (C=O) group at 1751 cm-1 in NS spectra confirmed the successful cross-linking of relative βCDs by DPC in various ratios(31). The 5-FU characteristic peaks were broadened or shifted in the formulations suggesting definite interactions between 5-FU and NS(16). The peaks correlated to the aromatic ring for the drug alone are weakened in the spectra of 5-FU loaded NS and some bands in the region between 2407 and 3100 cm-1 correlated to the aromatic ring result disappeared. These changes suggest the formation of the inclusion complexes(34),as shown in Fgure(7). Figure 4. FTIR of di-phenyl carbonate Figure 5:FTIR of -cyclodextrin Iraqi J Pharm Sci, Vol.29(2) 2020 5Fluorouracil nanosponges 95 Figure 6. FTIR of plain NS (1:4) Figure 7. FTIR of 5-FUNS Morphology studies AFM has been employed to Figure molecular structure of β-CD NS in the distilled water and examine their mechanical assets. The spherical crystalline NS presented the spectacular crystal planes with ordinary height of less than 400 nm. The SEM images of the plain βCD nanosponges were shown in Figure 8. SEM analysis revealed that nanosized particles with numerous pores on its surface. Figure 8. The AFM of βCD nanosponges. Iraqi J Pharm Sci, Vol.29(2) 2020 5Fluorouracil Nanosponges 96 Figure 9. The FE-SEM of βCD nanosponges In-vitro release study Drug release was performed in 0.1 N HCl. The 5-FU cumulative percent release of 5-FNS2 (Fig. 4a) showed a burst effect at the end of first hour. This fast release (56%) of the active ingredient was a result of increase in solubilization of the drug. After the first hour, the drug was released in a controlled manner indicating encapsulation of 5-FU in the nanostructures. 5-FU release from 5-FUNS2 was found to be higher than 5-FU-βCD complex(1:1) as compared to 5-FU solution as mentioned previously, this is belong to the carbonate linkage which was added to the primary hydroxyl groups of the parent CD unit. Thus, the drug molecules could be included inside the nanocavities of CD and due to the cross-linking further interactions of the guest molecules with more CD units might be thought. Moreover, the presence of the cross-linked network might also form nanochannels in the NS structure of the polymer mesh. This peculiar structural organization might be responsible for the increased solubilization and protection capacities of NS in comparison with the parent CD (16). Figure 10. 5-FU release from 5-FUNS2, 5-FUβCD complex and 5-FU solution in 0.1N HCl Conclusion Different molar ratios of (cyclodextrin to crosslinker) have a proficient effect on CE, Kst and entrapment efficiency of 5-FU. 5- FUNS2 with (1:4) molar ratio shows the best result of CE, Kst and entrapment efficiency. 5-FUNS2 gave higher release rate than 5-FU- βCD inclusion complex and 5-FU solution. Surface morphology of the prepared nanosponges by SEM, AFM and indicated nanosized and highly porous nanosponges. The overall results suggest that cyclodextrin nanosponge could be a promising 5-FU delivery system utilizing the suitable formula. 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