alginate beads of ketoprofen using experimental design: Formulation and in vitro dissolution evaluation

Accepted Manuscript Development of sericin/alginate beads of ketoprofen using experimental design: Formulation and in vitro dissolution evaluation

Emanuelle Dantas de Freitas, Paulo Cesar Pires Rosa, Meuris Gurgel Carlos da Silva, Melissa Gurgel Adeodato Vieira PII: DOI: Reference:

S0032-5910(18)30380-2 doi:10.1016/j.powtec.2018.05.016 PTEC 13389

To appear in:

Powder Technology

Received date: Revised date: Accepted date:

20 October 2017 28 March 2018 10 May 2018

Please cite this article as: Emanuelle Dantas de Freitas, Paulo Cesar Pires Rosa, Meuris Gurgel Carlos da Silva, Melissa Gurgel Adeodato Vieira , Development of sericin/alginate beads of ketoprofen using experimental design: Formulation and in vitro dissolution evaluation. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Ptec(2017), doi:10.1016/j.powtec.2018.05.016

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ACCEPTED MANUSCRIPT DEVELOPMENT OF SERICIN/ALGINATE BEADS OF KETOPROFEN USING EXPERIMENTAL DESIGN: FORMULATION AND IN VITRO DISSOLUTION EVALUATION Emanuelle Dantas de Freitasa, Paulo Cesar Pires Rosab, Meuris Gurgel Carlos da Silvaa,

Department of Processes and Products Design, School of Chemical Engineering, University

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Melissa Gurgel Adeodato Vieiraa*

of Campinas – UNICAMP, Albert Einstein Avenue, 500, 13083-852, Campinas, SP, Brazil Department of Pharmacology, School of Medical Sciences, University of Campinas –

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UNICAMP, Tessália Vieira de Camargo Street, 13083-887, Campinas, SP, Brazil *Corresponding author. E-mail address: [email protected]; Tel: +55 019 3521-3895.

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ABSTRACT - The development of new forms of drugs from the current existing active principles is an alternative to the costly and time-consuming process of developing new

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ones. The blend of sericin and alginate has been proposed as a polymer matrix for the incorporation of ketoprofen as a modified formulation. An experimental design was performed by varying the initial amount of alginate and drug added. Incorporation efficiency,

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drug loading, and release time of 85% were applied as response variables. Drug loading was statistically affected by both independent variables and the highest value (40.25 ± 0.23%) was

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achieved for the lower amount of alginate and higher amount of ketoprofen initially added. The incorporation efficiency was statistically affected only by the amount of alginate and its higher values were reached by the smaller amount of alginate added. Time to release 85% was not affected by any of the independent variables, although the longer times were achieved for the greater amount of drug added. In general, the best particle produced was K3, produced with the lowest amount of alginate and higher amount of ketoprofen initially added. The characterization analyzes of SEM, FTIR, XRD, OM and TGA confirmed the incorporation of ketoprofen. Keywords: Sericin; alginate; ketoprofen; modified release formulation. 1

ACCEPTED MANUSCRIPT 1. INTRODUCTION Ketoprofen is a propionic acid derivative drug, chemically 2-(3-benzoylphenyl) propionic acid. It is a nonsteroidal anti-inflammatory drug (NSAID), used as an antipyretic, analgesic, and anti-inflammatory in the treatment of rheumatoid arthritis and osteoarthritis [1]. Ketoprofen is known for its negative gastrointestinal effects and low plasma half-life, approximately 2 hours [2]. Considering the above, it is desirable to develop a modified release

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form for ketoprofen, in order to enhance its therapeutic effects and patient s’ compliance. One of the most widely used systems regarding drug modified release involves

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polymer matrix systems, defined as a well-mixed compound of a drug and a gelling agent as a

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hydrophilic polymer [3]. This matrix can be used as a beaded delivery formulation, wherein multiple beads containing the drug are encapsulated in a delivery capsule [4]. Several

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polymers can be applied for the incorporation of drugs, especially those biodegradable and biocompatible, of natural or synthetic nature[5] such as hydroxypropyl methylcellulose (HPMC) [6], hyaluronic acid [7], poly(D,L-lactic acid) (PLA) [8], poly(ethylene glycol)

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diamine [9].

In this context, sericin, one of the components of silk fiber, has been highlighted as a

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potential protein in the field of drug delivery [10]. Silk derived from the Bombyx mori

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silkworm is composed of 25 – 30% of sericin, which is known as “silk glue” and is usually discharged into wastewaters from the textile industry [11]. These wastewaters present high organic content and can deplete the oxygen dissolved in aquatic bodies. Thus, it is interesting

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to use this protein in a high added value activity [12]. Sericin is a globular protein composed of about 18 amino acids, mainly serine, with

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strong polar side chains, such as hydroxyl, carboxyl, and amino groups. Its structure is mainly involved in an amorphous random form, soluble in hot water and, to a lesser extent, an organized form in β-sheet [13]. In recent years, sericin has emerged as a viable resource due to its properties such as gelling ability, anti-bacterial, tyrosinase inhibitory action, antioxidant, and moisture absorption [14]. The chemical characteristics of sericin allow its crosslinking, blending, and polymerization with other polymers to improve its properties and, therefore, its applicability [11]. Among the possible polymers to form a blend with sericin, poly(vinyl alcohol) (PVA) [15], chitosan [16], keratin [17], and alginate [18] have been used.

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ACCEPTED MANUSCRIPT Alginate refers to salts of alginic acids, such as sodium alginate, which in commercial form is a natural polysaccharide extracted from brown algae [19]. Alginic acid consists of random or alternating arranged blocks of D-mannuronic acid (M) and L-guluronic acid (G), in which the M/G ratio strongly affects its physicochemical properties [20]. The alginate can easily cross-link with divalent ions, such as Ca2+, leading to the ionic gelation phenomenon. In this case, two adjacent carboxyl groups in the M or G unit bind to the divalent cation, forming the egg-box model, responsible for the sol-gel transformation. Due to this

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characteristic, alginate has been widely used for microencapsulation in the biomedical and pharmaceutical field, as in the case of protein [21], antibodies [22], vitamins [23], insulin

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[24], and drugs [25,26].

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In the context of drug modified release systems, the sericin-alginate blend has been proposed to improve the mechanical properties of the polymers, bio-adhesion, and drug

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release kinetics [27]. The aim of the present work was to produce drug-loaded sericin-alginate particles by the ionic gelation method, in order to obtain a gastro-resistant particle and

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modified release kinetics, using ketoprofen as the drug to be incorporated. Some studies involving ketoprofen-loaded alginate particles can be found in the literature [28,29]. However, in this work, we intended to evaluate and to improve drug particles by adding

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sericin, in addition to adding value to this protein. A 22 full factorial design with central points

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was performed to evaluate the effect of the process variables (amount of drug and amount of alginate added) on the percentage of drug incorporation, drug loading, and drug release kinetics.

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In the present work, a comparison of the dissolution of the obtained particles with the dissolution of the commercially available Profenid® Enteric drug reference was also

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performed. The drug in the commercial form has a wide range of excipients and, in the present work, it was intended to make a novel pharmaceutical form with natural bases and achieving results similar or superior to the market.

2. MATERIAL AND METHODS 2.1 Material As the source of sericin, silkworm (Bombyx mori) cocoons were provided by Bratac Silk Mills Company, based in Londrina – PR (Brazil). Commercial sodium alginate was supplied by Sigma-Aldrich (St Louis – MO, USA) and ketoprofen was obtained from 3

ACCEPTED MANUSCRIPT Purifarma (São Paulo – SP, Brazil). All reagents used were of analytical grade. Calcium chloride was purchased from AnidrolTM (Diadema – SP, Brazil), hydrochloric acid and sodium phosphate tribasic from DinâmicaTM (Diadema – SP, Brazil). Ultrapure water (Reverse osmosis, Gehaka, Brazil) was used to prepare all solutions. The Enteric Profenid® reference drug (Sanofi-Aventis, Brazil, L: 720227) was purchased from a local drugstore.

2.2 Extraction and cryo-concentration of sericin

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The cocoons of silkworms were cleaned and cut with the aid of tongs and scissors in order to remove any impurity and obtain small pieces (about 1 cm²). The small pieces were

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washed in water and rinsed with ultrapure water, and thus, dried in an oven at 50 ºC for 12

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hours. Sericin extraction was performed in an autoclave (AV-18, Phoenix, Brazil) following the method described by Silva et al. [18]. The cleaned and dried cocoons were immersed in

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ultrapure water in a ratio of 40:1 (grams of cocoons:liter of water). Sericin was extracted into the autoclave at 1 kgf/cm² and 120 ºC for 40 min and then stored in a tight container at room temperature for 12 hours to stabilize the hydrogel. Later, it was frozen and thawed at room

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temperature to obtain cryo-concentration of sericin. The solution was filtered and the retained sericin was heated in the autoclave at 1 kgf/cm² for about 10 min. Sericin concentration was

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adjusted to 2.5% (w/V) by dilution.

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2.3 Ketoprofen incorporation and preparation of particles Drug incorporation followed the methods described by Khandai et al. [30] and Vidart et al. [31]. 2.5% (w/V) sericin solution was autoclaved at 1 kgf/cm² for 10 min to solubilize

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the protein. Thus, the solution was stirred at 4000 rpm with an UltraturraxTM (T18, IKA, USA) to 55 ºC. Sodium alginate was then added to the solution until homogeneous.

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Subsequently, ketoprofen was added to the blend solution and dispersed with an UltraturraxTM, initially at 4000 rpm and then at 8000 rpm, until homogeneous. The preparation of particles followed the ionic gelation method, which is based on the cross-linking ability of the polyelectrolytes in the presence of multivalent cations and formation of hydrogel beads [32]. The drug loaded sericin/alginate solution was dripped using a peristaltic pump in a 3% (w/V) CaCl2 solution under continuous stirring. After dripping, the particles were shaken in a jar test at 100 rpm for 30 min, then washed with deionized water and dried at room temperature for at least 24 hours.

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ACCEPTED MANUSCRIPT 2.4 Ketoprofen calibration curve The standard calibration curve of ketoprofen was obtained in pH 6.8 phosphate buffer. A stock solution of ketoprofen of 0.2 g/L was prepared. From this, different solutions having concentrations in the range of 0.00125 – 0.0145 g/L were prepared. The solutions were analyzed by UV spectroscopy (UVmini1240, Shimadzu, Japan) at 258 nm.

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2.5 Experimental design The effect of the variables involved in particle preparation was evaluated using a 22

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full-factorial design consisting of factorial points at two levels and center point. The amount of sodium alginate added (X1) and the amount of drug added (X2) were chosen as

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independent variables and were classified as low, medium, and high values, as indicated in Table 1. The effects of the factors were analyzed on response variables, or dependent

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variables, namely drug encapsulation efficiency (Y1), drug loading (Y2) and time to release 85% (Y3). The design had four runs in triplicate of the center point to estimate the

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experimental error.

2.6 Drug loading and incorporation efficiency

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The ketoprofen incorporation efficiency was one of the response variables analyzed.

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To determine this parameter, 0.1 g of particles were immersed in 500 mL of phosphate buffer (pH 6.8), which is known to dissolve sericin/alginate blend [31], for about 24 hours. Thus, the dispersion was sonicated for 15 min (LS-9,5DA, LimpSonic, Brazil) and filtered using a 0.45

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µm filter. The ketoprofen concentration of the filtrate was determined by spectrophotometer at 258 nm. The analysis was performed in triplicate. The drug incorporation efficiency was

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estimated by Eq. 1 [33].

𝐼𝑛𝑐𝑜𝑟𝑝𝑜𝑟𝑎𝑡𝑖𝑜𝑛 𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑐𝑦 =

𝐸𝑥𝑝𝑒𝑟𝑖𝑚𝑒𝑛𝑡𝑎𝑙 𝑑𝑟𝑢𝑔 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 .100 𝑇ℎ𝑒𝑜𝑟𝑒𝑡𝑖𝑐𝑎𝑙 𝑑𝑟𝑢𝑔 𝑐𝑜𝑛𝑡𝑒𝑛𝑡

(1)

Where the Experimental drug content is obtained from the assay described above and the Theoretical drug content is calculated considering the fraction of drug added during the synthesis of particle. From the same assays, ketoprofen loading could be determined according to Eq. 2.

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𝐷𝑟𝑢𝑔 𝑙𝑜𝑎𝑑𝑖𝑛𝑔 =

𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑑𝑟𝑢𝑔 𝑖𝑛𝑐𝑜𝑟𝑝𝑜𝑟𝑎𝑡𝑒𝑑 .100 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑝𝑎𝑟𝑡𝑖𝑐𝑙𝑒𝑠

(1)

2.7 In vitro drug release – dissolution studies Dissolution studies allow the simulation of the gastrointestinal environment in order to assess the drug release over the time [34]. The blend used to produce the ketoprofen particles

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was the same as that used by Vidart et al. [35], who evaluated the incorporation of diclofenac sodium into sericin and alginate blend. Those authors confirmed the gastro-resistant

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characteristic of the particles, especially due to the presence of alginate. A similar result was observed by Tous et al. [29] for the incorporation of ketoprofen into alginate beads. Given

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the gastro-resistant characteristic of the blend used, in the present study, the ketoprofen dissolution assay was based on the method specified in U. S. Pharmacopeia (USP) XXXV

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[36] using a buffer stage to simulate enteric media. The amount of particles equivalent to 100 mg of drug content was added to the basket in dissolution equipment (UDT-814, Logan,

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USA) and contacted with 900 mL of phosphate buffer (pH 6.8) at 50 rpm and 37 ± 0.5 ºC for 8 hours to ensure the equilibrium condition. At predetermined times, 5 mL aliquots were collected, while 5 mL of fresh medium was replaced. The aliquots were filtered and the drug

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concentration again determined by spectrophotometry. Similar experimental conditions were

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used to evaluate the dissolution of the Enteric Profenid® reference drug.

2.8 Kinetic modelling of drug release

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To analyze the mechanism of release of ketoprofen, several models were applied to adjust experimental data, using Maple 17TM software. The adjusted coefficient of

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determination (R2adj) and the Akaike information criterion (AIC) were calculated to determine the best fit. The models are described below [37,38]. 2.7.1 Zero order model The zero order model describes systems in which the release of drug does not depend on its concentration and therefore the concentration is constant over time. Eq. 2 can describe this model. 𝑄 = 𝑄0 + 𝐾0 𝑡

(2)

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ACCEPTED MANUSCRIPT Where Q is the release of the drug at time t, Q0 is the initial amount of drug in solution and K0 is the zero order constant. 2.7.2 First order model The first order kinetic model describes the conventional drug release and considers the

ln 𝑄 = ln 𝑄0 − 𝐾1 𝑡

(4)

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(3)

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𝑑𝑄 = −𝐾1 . 𝑄 𝑑𝑡

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release exponentially over time, according to Eqs. 3 and 4.

Where K1 is the first order constant. This model is usually applied to describe the

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absorption or elimination of drugs. The mechanism on the theoretical basis is difficult to understand.

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2.7.3 Higuchi model

Higuchi proposed the first mathematical model to describe the release of drugs from a

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matrix system. The hypothesis assumed by Higuchi involves the initial concentration of drug in the matrix much larger than its solubility; occurrence of drug diffusion in only one

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dimension; insignificant swelling and dissolution of the matrix; and constant diffusivity of the drug. This model can be well applied to describe various types of modified drug release

(5)

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𝑄 = 𝐾𝐻 . 𝑡1/2

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forms, according to Eq. 5, the simplified Higuchi model.

In which KH is the Higuchi dissolution constant. Diffusion of drugs through the matrix is considered the rate-limiting step. 2.7.4 Weibull model The Weibull model can be applied to a wide range of dissolution curves, and according to Eq. 6, provides fraction accumulation of drugs in solution.

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𝑄 = 𝑄0 [1 − exp [

−(𝑡 − 𝑇)𝑏 ]] 𝑎

(6)

Where T is the lag time resulting from the dissolution process, the parameter a expresses the time dependence, and the parameter b describes the shape of the dissolution curve. The Weibull model is used to compare different matrix release profiles.

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2.7.5 Korsmeyer-Peppas model The Korsmeyer-Peppas model was developed to describe the release of drugs from

𝑄 = 𝐾𝐾𝑃 . 𝑡 𝑛 𝑄∞

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(7)

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polymer systems, according to Eq. 7.

𝑄

In which 𝑄 is the drug fraction released at time t, KKP is the rate constant, and ∞

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parameter n is associated with the drug delivery mechanism. Only data of up to 60% of drug release data should be adjusted by this model to determine the mechanism involved,

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according to Table 2, for cylindrical and spherical shape [39].

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In this case, drug diffusion may obey the Fick Law or may occur through gel swelling/relaxation (Case II transport) [40]. 2.7.6 Hopfenberg model

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The Hopfenberg model was developed to describe the release of drugs from polymers with erosive surface, considering the surface area remaining constant during the process.

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Thus, the actual erosion process is considered the rate-limiting step. Eq. 8 represents the model above.

𝑄 𝑘. 𝑡 𝑛 = 1 − [1 − ] 𝑄∞ 𝐶0 . 𝑎0

(8)

Where 𝑄∞ is the total amount release at exhaustion, k is the erosion rate constant, C0 is the initial drug concentration in the matrix, and 𝑎0 is the initial radius of a sphere or cylinder. The parameter n must be considered 2 for cylinder and 3 for sphere [41].

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ACCEPTED MANUSCRIPT 2.9 Characterization of the particles Drug particles were characterized to evaluate the incorporation of ketoprofen. All techniques performed are described in Table 3.

3. RESULTS AND DISCUSSION 3.1 Experimental design - drug loading, incorporation efficiency and

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drug release

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Drug loading expresses the amount of ketoprofen present in the particles produced while the drug incorporation efficiency refers to the amount of ketoprofen in the particles as a

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function of the amount initially added during the production of particles, i.e., the incorporation efficiency describes the efficiency of the preparation method to for

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incorporating drug into the polymer matrix. Both depend on the interactions between the drug, the matrix, and the surrounding medium [42]. The results obtained from the

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experimental design are shown in Table 4 and the three-dimensional response surface for all response variables are shown in Figure 1.

It is possible to observe the drug loading in the range of 20.54 to 40.25% for all

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formulations evaluated. This parameter was dependent on the production process, increasing

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as the amount of drug initially added increased and reducing when the amount of alginate increased. It is important to achieve high levels of drug loading to achieve the sufficiently high drug level per particle required for the incorporated drug to be therapeutically effective

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[43]. Statistically, both the amount of ketoprofen and alginate showed influence on drug loading with 90% confidence level.

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The drug incorporation efficiency showed values in the range of 74.96 to 91.19% for the formulations produced. Statistically, only alginate showed influence on the incorporation efficiency with 90% confidence level. The parameter showed the highest values when the alginate initially added increased. It is important to evaluate this parameter to avoid the loss of raw material during the production process. The last response variable analyzed was the time to 85% drug release. From Table 4, this parameter ranged from 92.36 ± 2.75 to 111.65 ± 11.50 min. Statistically, none of the independent variables had an influence on time for 85% drug release. However, from Figure

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ACCEPTED MANUSCRIPT 1, the amount of ketoprofen initially added showed influence on this response variable. The longer release time occurred for the formulations with higher amounts of drug initially added. To evaluate the effect of sericin on the particles, drug-loaded alginate particles were produced. The K2 particle, the worst case assessed in terms of drug incorporation efficiency, was chosen as the composition for comparison. Thus, particles with 2.0 g/L ketoprofen and 2.8 g/L sodium alginate, named K2*, were developed, and their results can also be found in

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Table 4. It is possible to observe that the addition of sericin to the particle is able to improve the drug incorporation efficiency, evidencing its importance. Possibly this was due to the

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strong polar side groups of sericin, such as hydroxyl, carboxyl, and amino groups [11]. During the ionic gelation step, such groups strongly cross-link alginate molecules, preventing

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loss of drug crystals prior to complete incorporation. In the case of alginate and drug compound particles, this strong cross-linking is not observed and more drug crystals are lost

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prior to entrapment. The time to release 85% of the drug was higher for K2*, showing that alginate is responsible for delaying drug release, although it has not been observed for

3.2 In vitro drug release

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alginate and sericin blend.

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In vitro release of ketoprofen was performed at in buffer solution (pH 6.8), simulating

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enteric media. The dissolution profiles obtained at in this step are shown in Figure 2 and present the data obtained up to 480 min, which were performed to guarantee the equilibrium condition.

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It can be seen that the drug release took about 3 hours to reach its maximum, regardless of the experimental design formulation. Shohin et al. [44] reviewed the

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monographs of ketoprofen for immediate-release solid oral dosage forms and concluded that 85% of the drug should dissolve in 30 min or less at pH 6.8. Thus, sericin/alginate particles loaded with ketoprofen were shown to be a modified release form, prolonging the drug release for up to 2 hours (85% of the drug) at pH 6.8, improving its therapeutic efficacy. For comparison purposes, the dissolution data of the K1-K4 formulations and the commercial Enteric Profenid® are shown in Figure 3. Such data were truncated to 180 min, which was the necessary equilibration time. It can be seen that the formulations developed showed a slower release than the reference drug. In addition, the reference drug reaches a lower final drug release than the particles developed in the present study, demonstrating the 10

ACCEPTED MANUSCRIPT greater availability of the drug for therapeutic action. Such difference can be confirmed by the analysis of the similarity factor (f2) (Table 5) between the formulations obtained and the commercial formulation. According to the FDA, a value of f2 > 50 indicates sufficient similarity between the dissolution profiles [45]. In the case of the present study, all formulations presented values lower than 50. Thus, a new release form was obtained, when compared to the commercial Enteric Profenid®.

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The formulation K2*, composed of alginate and ketoprofen, showed a slightly slower release when compared to formulations containing sericin. Nevertheless, it had lower release

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of drug at equilibrium, of about 90%. The formulations containing sericin and alginate blend

3.3 Kinetic modelling of drug release

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presented equilibrium release higher than 95%.

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The drug release profiles of Figure 2 were adjusted by the models of zero order, first order, Weibull, Higuchi, Korsmeyer-Peppas, and Hopfenberg for cylindrical and spherical

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forms. The parameters obtained for each model, the adjusted coefficient of determination (Radj²), and the Akaike Information Criterion (AIC) are shown in Table 6. From Table 6, it can be observed that the Weibull model presented the best fit for

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experimental data according to the highest R²adj values and the lowest AIC values. The

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Weibull model is a general empirical equation successfully applied to various types of dissolution curves. Because it is empirical, it is not appropriate to characterize the kinetic properties of dissolution [46], although it can describe the dissolution curves in terms of

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applicable parameters. In this case, the b value is the shape parameter, which characterizes the curve as an S-shape for all formulations evaluated (b > 1). From the parameters a and b, the

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time to release 63.2% of the drug (Td) could also be calculated and presented similar values for all the formulations [47]. The Hopfenberg model presented reasonable adjustments for both spherical and cylindrical systems, the latter being better. It is a mathematical equation based on the release of drugs from erosive surface devices. Thus, the rate-limiting step can be considered erosion of the matrix. From the Korsmeyer-Peppas model, a semi-empirical model, the release mechanism can be well evaluated [46]. For all formulations n > 0.89 and n > 0.85, which means that the drug release mechanism is a super case II transport, for cylindrical or spherical shape, respectively, involving polymer dissolution and chain disaggregation [37]. 11

ACCEPTED MANUSCRIPT The worst adjustments were observed for the Higuchi model. This is a theoretical model that describes the drug release as a diffusion process, based on Fick’s law. Thus, confirming the result of the Korsmeyer-Peppas model, diffusion does not contribute to the release of ketoprofen [46].

3.4 Characterization of drug particles

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3.5.1 Morphology Analysis (SEM) To evaluate the particle morphology SEM was performed and the micrographs

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obtained for the K1 – K4 formulations, the pure drug and the drug-free sericin/alginate blend

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are presented in Figure 4.

Particle micrographs with higher magnification (3,000x for the blend and 5,000x for

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the formulations) were taken from the cross-section. An increase in particle roughness can be observed due to drug incorporation. The surface of the particle composed of sericin and alginate blend is slightly smoother. The drug can be seen in the cross-section of the particles

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(K1(C), K2(C), K3(C) and K4(C)) and the higher amount of drug added has contributed to the sphericity and homogeneity of the particles when comparing K1(A) and K2(A) to K3(A) and

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K4 (A).

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3.5.2 Particle Size Analysis (OM)

Through optical microscopy (OM), images of the particles of each formulation were obtained. The 500 particle diameters were measured and the size distribution for each

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formulation (K1 – K7) is shown in Figure 5. The relative frequencies of the size distribution were adjusted by the Gauss fitting (normal) and the fit was analyzed by the adjusted

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coefficient of determination (R2adj). The values of R²adj and the mean diameter obtained for all formulations are exposed in Table 7. The particles containing ketoprofen had a diameter in the range of 1.75 ± 0.20 to 2.15 ± 0.24 mm. The increase in the initially added amounts of alginate and drug contributed to the increase of particle diameters. This may be associated with increased viscosity due to the higher amount of polymer. More binding sites become available, increasing cross-linking and thus avoiding the separation of coacervation droplets and favoring coalescence. Particle size was not related to the incorporation efficiency. However, the larger particles had the highest times for the release of 85% of the drug. 12

ACCEPTED MANUSCRIPT The Gauss adjustment provided adjusted coefficients of determination shown in Table 7. All formulations followed the normal distribution due to the high R²adj values, although formulation K1 shows the lowest value for this parameter. This means that all formulations have good reproducibility. 3.5.3 Cristallinity Analysis (XRD) Figure 6 shows the diffractograms obtained by X-ray diffraction for the formulations

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K1 – K7, pure ketoprofen, and sericin/alginate particles.

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It is expected that the sericin diffractogram shows a peak at 2θ = 20.5º, indicative of its β-sheet structure [48]. This can be observed in Figure 5 (SerAlg), evidencing the presence

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of sericin and confirming its structure did not change during the formation of the blend. The alginate diffractogram has two significant peaks at 13.4º and 23º [49]. They can also be

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observed in Figure 6 (SerAlg) confirming the presence of alginate in the blend. All peaks obtained for sericin/alginate particles are broad, demonstrating the amorphous characteristic

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of this sample.

The ketoprofen (Keto) diffractogram shows sharp diffraction peaks at 2θ = 13.13, 14.38, 17.31, 18.37, 20.07, 22.86, and 23.88º, which indicates that this is a crystalline solid

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[50]. Although some of these peaks can be observed in the diffractograms of the K1 – K7

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formulations, they present a larger and more diffuse pattern, probably due to the random arrangement of the molecule in the polymer matrix. Thus, it can be concluded that

ketoprofen.

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incorporation of the drug into sericin and alginate blend resulted in a more amorphous form of

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3.5.4 Functional groups (FTIR) The interaction between ketoprofen and sericin/alginate blend was evaluated by Fourier transform infrared (FTIR) analysis. Figure 7 shows the FTIR spectra obtained for the formulations K1 – K7, sericin/alginate particle, and pure ketoprofen. The FTIR spectrum from ketoprofen showed characteristic absorption peaks at 1696 and 1651 cm-1, denoting C=O stretching of carboxylic acid and ketone, respectively. The absorption peaks at 1601 and 1440 cm-1 were due C=C and C-C stretching from the aromatic ring, respectively [51]. Regarding the K1 – K7 formulations, similar peaks have been observed especially due to acidic and ketonic carbonyl groups. There was no displacement or 13

ACCEPTED MANUSCRIPT disappearance of the characteristic bands, indicating that there is no strong interaction between the drug and the polymers used and, thus, confirming their compatibility. 3.5.5 Thermal Analysis (TG/DTG and DTA) Thermal curves for ketoprofen, K1 – K7 formulations and blank particles (without drug) were obtained in a dynamic nitrogen atmosphere. Figure 8 shows the curves obtained

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for ketoprofen (Keto), sericin/alginate particle (SerAlg), and K1 – K4 formulations. The ketoprofen (Keto) DTA curve shows an endothermic event between 89.67 and

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136.02 ºC indicating drug melting, which usually occurs in the range of 94 – 97 ºC. The TG/DTG curves did not show loss of mass in this temperature range. The ketoprofen

SC

decomposition appeared to occur in two different steps. First, an endothermic event was observed in the DTA curve between 214 and 397 ºC. This is related to the thermal

NU

decomposition process, confirmed by the TG/DTG curves, which presented a strong mass loss in this temperature range, with a peak at 314.85 ºC. The second step was the exothermic event

MA

between 503.64 and 555.95 ºC, attributed to the oxidation of organic matter. Combining TG/DTG and DTA curves, it is verified that the stability of ketoprofen is up to 214 ºC [52].

D

Sericin extracted from Bombyx mori generally exhibits a pattern in its thermal analyzes with two endothermic events near 212 and 319 ºC. The first is related to the

PT E

molecular movement and the melting of peptide bonds and the latter is related to the thermal decomposition [53]. From Figure 8 (SerAlg), equivalent endothermic events were observed at about 216 and 412 ºC, evidencing that the blend achieved significantly greater thermal

CE

stability due to cross-linking and increasing molecular weight. Alginate also exhibits a pattern in its thermal analyze. It is usually decomposed by dehydration, followed by degradation to in

AC

Na2CO3 and a carbonized material in the temperature range of 202.8 – 585 ºC. Figure 8 (SerAlg) an intense exothermic event around 600 ºC due to the decomposition of the carbonized material in Na2CO3 [54]. The DTA curves of the sericin/alginate particles and the K1 – K4 formulations showed endothermic peaks around 100 ºC, mainly due to the evaporation of the water. These peaks in the formulations were more pronounced, probably due to the simultaneous melting of ketoprofen at this temperature. The TG/DTG curves of the K1 – K4 formulations showed a significant mass loss around 300 ºC due to the degradation of ketoprofen and the decomposition of sericin. Furthermore, the loss of mass in the range of 170 – 370 ºC may also 14

ACCEPTED MANUSCRIPT refer to the destruction of glycosidic bonds [55]. At 600 ºC, the intense exothermic event evidences the formation of Na2CO3 as a result of the alginate decomposition. Figure 9 shows all TG curves obtained for the sericin/alginate particle, K1 – K4 formulations and ketoprofen as a function of the percentage of mass loss. It can be observed that the formulations K1 – K4 presented loss profile of mass similar to the drug-free blend, whereas pure ketoprofen presented a strong loss of mass after 214 ºC. Thus, it can be concluded that the drug

PT

incorporated into the blend has become more stable.

RI

4. CONCLUSIONS

The blend of sericin and alginate was successfully applied for the incorporation of

SC

ketoprofen. When comparing the particles with and without sericin, it is observed that this protein improved the incorporation of the drug. Statistically, in relation to the

NU

sericin/alginate/drug composite particles, drug loading was affected by the amounts of alginate and drug initially added, and the incorporation efficiency was affected only by the

MA

amount of alginate initially added. The release time of 85% was not statistically affected by any of the independent variables analyzed. The formulations were analyzed by the highest drug loading values, incorporation efficiency, and time to release 85%. Thus, the best

D

formulation was K3, composed of the lower amount of alginate and the greater amount of

PT E

ketoprofen. Comparison of the drug release profile of the particles to with the reference drug Enteric Profenid® showed similar behavior. The particles released the drug more slowly and achieved a higher drug release value than Enteric Profenid®. SEM analysis showed an

CE

increase in particle roughness due to the presence of drug. The FTIR spectra proved the compatibility between the drug and the blend used, since no strong interaction occurred. XRD

AC

analysis showed that incorporation of the drug into the sericin/alginate blend resulted in a more amorphous form of ketoprofen. OM analysis demonstrated the increase in particles size due to the increases amounts of alginate and drug initially added, as well as the good reproducibility of the particles. Thermal analysis showed that ketoprofen was thermally more stable after incorporation into the blend.

Acknowledgements The authors acknowledge the financial support received from CNPq (Proc. 470615/2013-3 and 300986/2013-0) and FAPESP (Proc. 2015/13505-9 and 2016/05007-1) and the supply of cocoons by BRATAC Company. 15

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[54] J.P. Soares, J.E. Santos, G.O. Chierice, E.T.G. Cavalheiro, Thermal behavior of alginic acid and its sodium salt, Eclética Química. 29 (2004) 57–64. doi:10.1590/S0100-

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AC

CE

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Biomater. 5 (2016) 117–133. doi:10.1007/s40204-016-0051-9.

21

ACCEPTED MANUSCRIPT Table 1 – Factors and their levels for Full-Factorial Design. Factor level Factor 0

+1

% Sodium Alginate (w/V)¹

2.0

2.4

2.8

% Ketoprofen (w/V)¹

2.0

3.0

4.0

AC

CE

PT E

D

MA

NU

SC

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¹Concentration in drug loaded sericin/alginate solutions

PT

-1

22

ACCEPTED MANUSCRIPT Table 2 – Diffusion exponent and mechanism of drug release for cylindrical and spherical shape. Diffusion exponent (n) Mechanism of drug release Spherical shape

0.45

0.43

Fickian diffusion

0.45 < n < 0.89

0.43 < n < 0.85

Non-Fickian diffusion

0.89

0.85

Case-II transport

n > 0.89

n > 0.85

RI

PT

Cylindrical shape

AC

CE

PT E

D

MA

NU

SC

Super case-II transport

23

ACCEPTED MANUSCRIPT Table 3 – Characterization analyses of ketoprofen and drug particles. Analysis

Optical Microscopy (OM)

Equipment

Parameters

National Stereo Microscope,

500 particles diameters were

model DC4-456H

measured

LEO Electron Microscopy,

Microscopy (SEM)

temperature and coated with

model LEO 440i

PT

Particles dried at room

Scanning Electron

a layer of gold

RI

Cu Kα adsorption, 40 kV,

X-ray diffraction (XRD)

Fourier transformed

NU

SC

Philips, model X’Pert-MPD

Thermo Scientific, model

infrared spectroscopy

Nicolet 6700

MA

(FTIR) Thermal Analysis

Shimadzu, model DTG-60

0.02º, scan speed: 0.02º/s, angle of incidence: 5 – 50º Sample analyzed as KBr pastille form in the range 4000 – 400 cm-1 N2 outflow: 50 mL/min, heating rate: 20 ºC/min, temperature range: 30 – 1000 ºC

AC

CE

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D

(TGA/DTG and DTA)

current: 40 mA, step size

24

ACCEPTED MANUSCRIPT Table 4 – Results obtained from experimental design. Ketoprofen Incorporation

Drug loading

(%,w/V)

(%,w/V)

efficiency (%)

(%)

K1

2.0

2.0

91.19 ± 0.41

28.06 ± 0.13

94.15 ± 1.57

K2

2.8

2.0

74.96 ± 0.55

20.54 ± 0.15

92.36 ± 2.75

K3

2.0

4.0

85.53 ± 0.48

40.25 ± 0.23

103.73 ± 2.80

K4

2.8

4.0

76.48 ± 2.69

32.90 ± 1.16

105.09 ± 1.36

Central points

2.4

3.0

83.87 ± 6.33

31.85 ± 2.40

111.65 ± 11.50

K2*

2.8

2.0

66.07 ± 0.75

SC

RI

PT

Alginate

25.09 ± 0.28

t85 (min)

158.33 ± 38.87

AC

CE

PT E

D

MA

NU

Formulation

25

ACCEPTED MANUSCRIPT Table 5 – In vitro similarity factor (f2) for comparison of the dissolution profile of the obtained formulations and the Enteric Profenid®. Alginate (%,w/V)

Ketoprofen (%,w/V)

f2

K1

2.0

2.0

48.35

K2

2.8

2.0

46.22

K3

2.0

4.0

K4

2.8

4.0

Central points

2.4

3.0

K2*

2.8

2.0

38.74 37.98 40.07 38.37

AC

CE

PT E

D

MA

NU

SC

RI

PT

Formulation

26

ACCEPTED MANUSCRIPT Table 6 – Parameters obtained from drug release modelling of K1 – K7 formulations. Formulation K3

K4

K5

K6

K7

0.888

0.883

0.818

0.804

0.648

0.620

0.871

R2adj

0.865

0.877

0.945

0.928

0.789

0.901

0.901

AIC

53.15

52.24

44.08

46.09

67.83

59.19

50.08

0.015

0.014

0.013

0.012

0.014

0.011

0.014

R2adj

0.750

0.679

0.742

0.688

0.896

0.806

0.756

AIC

54.63

57.00

53.89

55.65

56.06

61.46

54.11

a

1631.06

3960.79

1473.91

2641.21

423.63

1614.47

1310.41

b

1.849

2.063

1.747

1.883

1.455

1.702

1.770

Td

54.63

55.46

65.11

65.65

63.90

76.71

57.71

R2adj

0.992

0.998

0.993

0.998

0.987

0.998

0.996

30.84

19.77

28.84

18.92

41.05

24.47

24.99

7.22

7.06

6.52

6.32

6.87

6.32

7.03

R2adj

0.408

0.342

0.362

0.306

0.644

0.503

0.400

AIC

61.98

63.67

61.74

63.10

66.81

70.24

61.85

1.531

1.111

0.870

0.663

2.724

1.206

1.298

Ordem-zero

Weibull

AIC

PT E

Primeira-ordem

D

𝐾1

AC

Higuchi

CE

𝐾𝐻

𝐾𝐾𝑃

RI

𝐾0

PT

K2

NU

K1

SC

Parameter

MA

Model

Korsmeyer-

n

1.478

1.609

1.419

1.534

1.338

1.267

1.438

Peppas

R2adj

0.848

0.853

0.935

0.922

0.841

0.900

0.888

AIC

53.74

53.99

46.05

47.82

62.54

58.87

51.10

0.0062

0.0060

0.0053

0.0051

0.0056

0.0045

0.0059

Hopfenberg; n = 2 k2

27

ACCEPTED MANUSCRIPT 0.885

0.840

0.893

0.848

0.954

0.934

0.898

AIC

49.52

52.28

47.75

50.59

50.65

53.16

48.24

k3

0.0044

0.0042

0.0038

0.0036

0.0040

0.0033

0.0042

Hopfenberg; n = 3 R2adj

0.849

0.792

0.848

0.798

0.945

0.900

0.858

AIC

51.26

54.00

50.17

52.55

51.53

56.29

50.39

AC

CE

PT E

D

MA

NU

SC

RI

PT

R2adj

28

ACCEPTED MANUSCRIPT Table 7 – Mean particle diameters and R²adj from Gauss fitting for formulations K1 – K7. Mean diameter (mm)

R²adj

K1

1.75 ± 0.20

0.843

K2

1.92 ± 0.20

0.968

K3

2.08 ± 0.23

0.918

K4

2.15 ± 0.24

0.930

K5

2.15 ± 0.22

0.882

K6

2.05 ± 0.21

K7

2.07 ± 0.22

RI

PT

Formulation

SC

0.949

AC

CE

PT E

D

MA

NU

0.940

29

ACCEPTED MANUSCRIPT Figure 1 – Three-dimensional response surface for the response variables (a) drug loading, (b) incorporation efficiency, and (c) time for 85% drug release. Figure 2 – Dissolution profiles obtained for K1 – K4 formulations, central points of experimental design and K2* formulation. Figure 3 – Comparison between dissolution profiles obtained for K1 – K4 formulations and commercial Enteric Profenid®.

PT

Figure 4 – Micrographs obtained from pure drug (Keto), sericin/alginate blend (Ser/Alg), and K1 – K4 formulations at (A) magnification of 150x, (B) magnification of 3,000x, and (C)

RI

magnification of 5,000x.

SC

Figure 5 – Size distribution obtained for K1 – K7 formulations.

Figure 6 – X-ray diffractograms of K1 – K7 formulations, pure ketoprofen, and

NU

sericin/alginate blend.

Figure 7 – FTIR spectra from sericin/alginate blend (Ser+Alg), pure ketoprofen (Keto) and

MA

K1 – K7 formulations.

Figure 8 – TG/DTG and DTA curves for sericin/alginate blend (SerAlg), pure drug (Keto),

D

and K1 – K4 formulations.

Figure 9 – TG curves as the percentage of mass loss for sericin/alginate blend (SerAlg), pure

AC

CE

PT E

drug (Ketoprofen), and K1 – K4 formulations.

30

ACCEPTED MANUSCRIPT Highlights Ketoprofen was incorporated into sericin/alginate blend through statistical design.



Sericin improved drug incorporation about 13.5%.



Low amount of alginate and large amount of drug provided the lowest time to release.



Drug release time of the particles was 4 times slower than immediate-release form.



The main mechanism for drug release was matrix erosion.

AC

CE

PT E

D

MA

NU

SC

RI

PT



31

Graphics Abstract

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9