CONTROLLED RELEASED BEADS: DEVELOPMENT, ADVANCEMENT AND ASSESSMENT OF ANTIMICROBIAL DRUGS

Abstract

The present invention relates to a method for preparing controlled-release chitosan-sodium alginate microbeads containing Levofloxacin Hydrochloride. The method involves preparing a sodium alginate solution, incorporating Levofloxacin Hydrochloride, and adding it dropwise into a chitosan-calcium chloride solution to form microbeads via ionic gelation. The microbeads are then cured, filtered, washed, and dried. The process allows for the formation of beads with varying sodium alginate and chitosan concentrations, which are suitable for controlled drug release applications. The resulting microbeads exhibit a prolonged release profile, with a drug release ranging from 69.88% to 82.13%, a yield of 74.07% to 83.58%, and a particle size range of 1.15 mm to 1.37 mm. These beads are biologically compatible, non-toxic, and exhibit enhanced antibacterial activity, making them suitable for antimicrobial drug delivery systems. Furthermore, the method enables efficient drug entrapment, with a drug entrapment percentage ranging from 35% to 67%.

Specification

Description:FIELD OF INVENTION
The present invention relates to the field of medicinal chemistry and organic synthesis, particularly the synthesis of controlled released beads, their advancement and assessment of antimicrobial drugs.

BACKGROUND
The most desirable and convenient method of drug administration is the oral route due to the ease of administration and patient compliance. A number of approaches will be developing to increase the residence time of dug formulation. One of the approaches the formulation of gastro retentive dosage forms in the form of muco-adhesive microspheres. Microsphere carrier systems, made from natural polymers are attracting considerable attentions for several years, for sustained drug delivery. Today, those dosage forms which can control the release rates and which are target specific have a great impact in development of novel drug delivery systems. These microspheres provide advantages such as efficient absorption and increased bioavailability of drugs owing to high surface-to-volume ratio, a much more intimate contact with the mucus layer and specific targeting of drugs to the absorption site. The applications of nanoparticles distributed in an organic matrix are: catalysis, magnetics, and photonics. The nanoparticle loaded polymer coating on colloidal particles is desired as colloidal particles can offer more surface area which is advantageous for a wide range of applications, such as catalysis and sensor applications. Oral delivery of drugs is by far the most preferred route of drug delivery due to ease of administration, patient compliance and flexibility in formulation. The design of oral controlled drug delivery systems (DDS) is primarily aimed to achieve more predictable and increased bioavailability. Various types of drug delivery systems for oral administration such as drug release rate-controlled delivery systems, time-controlled delivery systems and site-specific delivery systems have been extensively developed. Controlled-release drug delivery systems (CRDDS) provide drug release at a predetermined, predictable, and controlled rate. An important requisite for the successful performance of oral CRDDS is that the drug should have good absorption throughout the gastrointestinal tract (GIT), preferably by passive diffusion, to ensure continuous absorption of the released drug.

The impact of antimicrobial agents on public health over the past 50 years is unmatched by any other therapeutic class. Precise data on current antibiotic use are difficult to obtain; in the UK, in the community alone, there are over 50 million prescriptions and over 250 million defined daily doses of antibiotics each year.

The term "microcapsule" is defined, as a spherical particle with the size varying between 50 nm to 2 mm containing a core substance. Microspheres are in strict sense, spherically empty particles. However, the terms microcapsules and microspheres are often used synonymously. In addition, some related terms are used as well. For example, "microbeads" and "beads" are used alternatively. Sphere and spherical particles are also employed for a large size and rigid morphology. Due to attractive properties and wider applications of microcapsules and microspheres, a survey of the applications in controlled drug release formulations is appropriate. Although the word capsule implies a core and shell structure, the term microcapsules admit not only membrane enclosed particles or droplets but also dispersion in solid matrix lacking a distinctive external wall phase as well as intermediate types.

Microcapsules are finally dispersed in various dosage forms, such as hard gelatin capsules, which may be enteric coated, soft gelatin capsules, or suspensions in liquids, all of which allow dispersion of individual microcapsules on release. Microcapsules continue to be of much interest in controlled release because of relative ease in design and formulation and partly on the advantages of microparticulate delivery systems.

Chitosan is a linear polysaccharide composed of randomly distributed ß-(1?4)-linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit). It is made by treating the chitin shells of shrimp and other crustaceans with an alkaline substance, like sodium hydroxide
US10537873B2 discloses the Synthesis and application of MOFs/natural polymers composite beads adsorbents. To overcome the drawback of MOFs, in an embodiment, both novel MOFs/sodium alginate (MOFs/SA) and MOFs/chitosan (MOFs/CS) composite beads were prepared and characterized. Each composite beads include one or more of MIL-101 (Cr), MIL-100 (Cr), MIL-53 (Al), MIL-100 (Al), NH2-MIL-101 (Al), UIO-66, ZIF-8, ZIF-68, ZIF-67, and ZIF-9-67 nanoparticles. Adsorption of anionic contaminants onto the two composite beads was investigated and compared with pristine sodium alginate beads (SA) and chitosan beads (CS). The novel MOFs/SA beads all exhibit much higher adsorption capacity than SA beads; the novel MOFs/CS beads all exhibit much higher adsorption capacity than CS beads, which indicates that MOFs played a key role in the adsorption of anionic contaminants. The porous composite beads disclosed herein and related methods and devices may be used in adsorption technologies.

US11839685B2 provides a composition of matter comprising liposomes encapsulating in their intraliposomal aqueous compartment at least one active agent, the liposomes having a diameter of at least 200 nm and being embedded in a water insoluble, water absorbed cross-linked polymeric matrix. In one embodiment, the composition of matter is held within an aqueous medium, preferably being in iso-osmotic equilibrium with the intraliposomal aqueous compartments of the liposomes. The present disclosure also provides a method of removal of non-encapsulated active agent from the composition of matter, a method of preparing said composition of matter, a pharmaceutical composition comprising said composition of matter, use of such composition of matter; a method of providing prolonged delivery of a active agent to a subject in need thereof by administering to said subject the composition of matter disclosed herein as well as a package comprising said composition of matter held within said aqueous medium and instructions for use thereof.

US20210015142A1 provides a method for preparing double-layered bursting beads with milk tea flavor comprising: preparing inner and/or outer shell forming solutions for inner and/or outer shells of the bursting beads; preparing inner and/or outer core material solutions for inner and/or outer core materials of the bursting beads; preparing inner and/or outer shell curing solutions for inner and/or outer shells of the bursting beads; adding the inner core material solution into the inner shell forming solution, incubating, curing in the inner shell curing solution, and filtering to obtain inner bursting beads; and adding the inner bursting beads and the outer core material solution into the outer shell forming solution, and curing in the outer shell curing solution to obtain the double-layered bursting beads. The present invention improves bursting ability, densification, product instability due to complex browning and precipitation between tea polyphenol and protein during storage, and mechanical properties thereof.

Recent advances in drug delivery systems focus on controlling the release rate of drugs and targeting their release to specific sites within the body. Controlled-release dosage forms are designed to deliver the active pharmaceutical ingredient (API) at a predetermined rate, which is crucial for drugs that require long-term or sustained therapeutic effects. The incorporation of chitosan, a naturally occurring polysaccharide derived from chitin, into such systems provides numerous benefits. Chitosan has been shown to enhance mucoadhesion, increase drug retention at the target site, and exhibit antimicrobial properties, making it a suitable polymer for the formulation of controlled-release systems.

Levofloxacin Hydrochloride, a broad-spectrum antibiotic, has been widely used to treat infections caused by various bacteria. However, the therapeutic effectiveness of Levofloxacin is limited by its short half-life and the need for frequent dosing. Developing a controlled-release formulation of Levofloxacin Hydrochloride is highly desirable to improve its bioavailability, reduce dosing frequency, and ensure a sustained antimicrobial effect.

The present invention seeks to address these challenges by providing a method for preparing chitosan-sodium alginate microbeads containing Levofloxacin Hydrochloride for controlled release applications. By utilizing ionic gelation techniques, these microbeads are designed to release the drug in a controlled manner over an extended period. The formulation offers several advantages, including enhanced drug bioavailability, improved antibacterial activity, reduced cytotoxicity, and the ability to deliver Levofloxacin to the target site over a prolonged period. This innovative system holds significant potential for the development of effective oral controlled-release drug delivery systems, particularly for the treatment of bacterial infections.

OBJECTS OF THE INVENTION
The present invention has been developed in response to the present state of the art, and in particular, in response to the problems and needs in the art that have not yet been fully solved by currently available techniques and processes.

Accordingly, the present invention has been developed to provide a controlled-release nano -formulation employing the natural biodegradable polymer sodium alginate and chitosan (CS) as the vehicle which is biologically compatible, nontoxic, and has excellent antibacterial qualities in order to increase its antimicrobial activity and decrease cytotoxicity.

Therefore, the current invention successfully overcoming all of the above-discussed shortcomings present in the art.

1. It is an object of the invention to provide a method for preparing controlled-release chitosan-sodium alginate microbeads containing Levofloxacin Hydrochloride, designed to deliver the drug at a sustained and predictable rate over an extended period, thereby improving therapeutic efficacy and reducing dosing frequency.
2. It is an object of the invention to optimize the formulation of chitosan-sodium alginate microbeads by varying the concentrations of sodium alginate (2%, 4%, or 6% w/v) and chitosan (0.25%, 0.50%, or 0.75% w/v), allowing for the controlled modulation of drug release rates to meet specific therapeutic needs.
3. It is an object of the invention to ensure the effective incorporation of Levofloxacin Hydrochloride into the microbeads, thereby enabling the sustained release of the drug at therapeutic levels, providing enhanced bioavailability and reducing side effects due to fluctuations in drug concentration.
4. It is an object of the invention to prepare biologically compatible and non-toxic microbeads that maintain their structural integrity and release profile over time, thereby ensuring their suitability for long-term drug delivery applications, especially in the treatment of bacterial infections.
5. It is an object of the invention to enhance the antibacterial properties of the chitosan-sodium alginate microbeads, leveraging the inherent antimicrobial activity of chitosan while minimizing cytotoxicity, thus improving the safety and efficacy of the drug delivery system.
6. It is an object of the invention to achieve a controlled release profile of Levofloxacin Hydrochloride, with the drug release ranging from 69.88% to 82.13%, ensuring the formulation provides the desired therapeutic effect over a sustained period.
7. It is an object of the invention to achieve a high yield in the preparation of chitosan-sodium alginate microbeads, with a percentage yield ranging from 74.07% to 83.58%, ensuring efficient production and scalability of the formulation for commercial use.
8. It is an object of the invention to control the particle size of the chitosan-sodium alginate microbeads within the range of 1.15 mm to 1.37 mm, optimizing the surface area for drug release and ensuring that the beads are suitable for oral administration.
9. It is an object of the invention to optimize drug entrapment within the microbeads, achieving a drug entrapment efficiency of 35% to 67%, which enhances the overall performance of the controlled-release system by ensuring a sufficient amount of drug is retained within the beads for prolonged release.
10. It is an object of the invention to provide a versatile and scalable method for the preparation of chitosan-sodium alginate microbeads that can be easily adapted for the delivery of various active pharmaceutical ingredients (APIs), particularly for controlled release applications in oral drug delivery systems.

How the foregoing objects are achieved will be clear from the following brief description. In this context, it is clarified that the description provided is non-limiting and is only by way of explanation. Other objects and advantages of the invention will become apparent as the foregoing description proceeds, taken together with the accompanying drawings and the appended claims.

SUMMARY
This summary is provided to introduce a selection of concepts in a simplified format that is further described in the detailed description of the invention. This summary is neither intended to identify key or essential inventive concepts of the invention and nor is it intended for determining the scope of the invention.

The present invention focuses on the development, advancement, and assessment of controlled-release beads containing antimicrobial drugs, specifically Levofloxacin Hydrochloride. The aim is to address the growing concerns over bacterial resistance and improve the therapeutic efficacy of antibiotics. The invention utilizes a natural biodegradable polymer system consisting of sodium alginate and chitosan, which are combined to form microbeads that exhibit excellent biological compatibility, nontoxicity, and enhanced antimicrobial properties.

The controlled-release beads are created using an ionic gelation technique, where sodium alginate solutions with varying concentrations (2%, 4%, or 6%) are prepared and mixed with Levofloxacin Hydrochloride. This solution is then added dropwise to a chitosan-calcium chloride solution, resulting in the formation of microbeads. These beads are cured, filtered, washed, and dried to ensure optimal drug entrapment and controlled release. The beads are evaluated for several parameters, including particle size, drug entrapment efficiency, in vitro drug release profile, and antimicrobial properties.

The formulated beads show promising results, with drug release ranging from 69.88% to 82.13% over an 8-hour period, depending on the concentration of sodium alginate and chitosan. The microbeads are characterized by a particle size range of 1.15 mm to 1.37 mm and drug entrapment efficiency of 35% to 67%. The optimization and validation of the formulations were performed using response surface methodology, ensuring consistency and predictability of the drug release profile.

Further, the beads were subjected to various characterization techniques, including Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and differential scanning calorimetry (DSC), confirming the stability and compatibility of the drug within the polymeric matrix. The in vitro release studies demonstrated that the microbeads provide a sustained release of Levofloxacin, making them suitable for prolonged antimicrobial treatment with reduced frequency of administration.

The invention offers a highly effective and stable system for oral drug delivery, specifically for antibiotics like Levofloxacin Hydrochloride, targeting enhanced bioavailability and improved patient compliance. The beads' antimicrobial properties, coupled with controlled release, help in overcoming bacterial resistance and improving treatment outcomes in infections.

To further clarify the advantages and features of the present invention, a more particular description of the invention will be rendered by reference to specific embodiments thereof, which is illustrated in the appended figures. It is appreciated that this figure depicts only typical embodiments of the invention and are therefore not to be considered limiting of its scope. The invention will be described and explained with additional specificity and detail with the accompanying figure.

BRIEF DESCRIPTION OF FIGURES
These and other features, aspects, and advantages of the present invention will become better understood when the following detailed description is read with reference to the accompanying figures in which like characters represent like parts throughout the figures, wherein:

Figure 1, illustrates a view of XRD of drug Levofloxacin for the present invention.

Further, skilled artisans will appreciate that elements in the figures are illustrated for simplicity and may not have been necessarily been drawn to scale. For example, the flowcharts illustrate the method in terms of the most prominent steps involved to help to improve understanding of aspects of the present invention. Furthermore, in terms of the construction of the device, one or more components of the device may have been represented in the figures by conventional symbols, and the figures may show only those specific details that are pertinent to understanding the embodiments of the present invention so as not to obscure the figures with details that will be readily apparent to those of ordinary skill in the art having benefit of the description herein.

DETAILED DESCRIPTION:
For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the figures and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated system, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates.

It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be restrictive thereof.

Reference throughout this specification to "an aspect", "another aspect" or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrase "in an embodiment", "in another embodiment" and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.

The terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process or method that comprises a list of steps does not include only those steps but may include other steps not expressly listed or inherent to such process or method. Similarly, one or more devices or systems or elements or structures or components proceeded by "comprises... a" does not, without more constraints, preclude the existence of other devices or other systems or other elements or other structures or other components or additional devices or additional systems or additional elements or additional structures or additional components.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The system, methods, and examples provided herein are illustrative only and not intended to be limiting.

The terms "a" and "an" herein do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced items.

The terms "having", "comprising", "including", and variations thereof signify the presence of a component.

Now the present invention will be described below in detail with reference to the following embodiment.

Example 1:
Extraction of alginate
The first step in the alginate extraction is acidification step in which milled algal tissue is extracted with 0.1-0.2 M mineral acid which results in an ion exchange with protons. The acidification step will also allow the removal of contaminant glycans, like laminar an and furan. The second step is alkaline extraction in which the alginic acid gets converted into water soluble sodium alginate by neutralization of solution with alkali such as sodium carbonate or sodium hydroxide.

Example 2:
Preformulation Study
Preformulation testing is the first step in the rational development of dosage forms of a drug substance. It can be defined as an investigation of physical and chemical properties of a drug substance alone and when combined with excipients. The Preformulation testing is to generate information useful to the formulator in developing stable and bioavailable dosage forms that can be mass produced.

Example 3:-
Procedure
• The drug material is added in to above solutions till Supersaturated Solution is from.
• The Mixture can place in Orbital Shaker for 24 hrs. After 24 hrs, mixture was filtered and absorbance was measured.
• To detect the Concentration of Drug is Soluble in Different Solutions.

Example 4:-
Calibration Curve of Levofloxacin HCl
Calibration Curve is determined by using UV Spectrophotometric methods. In which 10 mg drug is added in 100 ml of water (100 µg /ml Solution). Different Dilutions (0, 2, 4, 6, 8, 10, 12) of above solution (100 µg/ml Solution) were prepared and absorbance was noted in respective ? max 294 nm. (Table 1).
Table 1: Formulation of difference between of Levofloxacin

Batch Code Coded Value Actual Value
Sodium Alginate
(X1) Chitosan
(X2) Sodium Alginate
(mg) Chitosan
(mg)
F1 -1 1 3 0.77
F2 0 1 2 0.71
F3 1 1 5 0.70
F4 -1 0 1 0.45
F5 0 0 3 0.55

Example 5:
Preparation of beads
The chitosan sodium alginate beads were prepared by ionic gelation technique. Sodium alginate solution (2%,4% & 6% w/v) were prepared by dissolving 2, 4 & 6g of Sodium alginate in a small amount of distilled water to get clear solution and volume was made up to 100 mL. Levofloxacin Hydrochloride was added to each Sodium alginate solution with continuous stirring using magnetic stirrer. Chitosan (0.25, 0.50 & 0.75%) and calcium chloride (3%) were dissolved in 5% acetic acid solution. Levofloxacin HCl containing Sodium alginate solution was added drop wise through 21-gauge needle into chitosan containing calcium chloride solution (100 ml) to formulate beads. The formulated beads were kept for 30 m for curing rection and then microbeads were collected by filtration using Wittman filter paper and washed with water. The drying of beads was done till constant weight achieved.

Example 6:
Evaluation of prepared beads
Characterization of beads beads
Fourier-transform infrared (FTIR) spectra of formulated bead was plotted using a Jasco FTIR 6100 type A spectrometer (Japan the spectra were recorded for the sample of prepared beads in KBr disks over the wavenumber 4000-400 cm-1. The powder X-Ray diffraction pattern of Levofloxacin, Tucsonan DRC were taken by Philips PW 1729 X-ray diffractometer Legroupinterconnexion, Scient Jurie, Caldara. Radiation generated from copper source with 5x10-3 cycles / sec was used. A mettle Toledo differential scanning colorimeter 821 (mettle Toledo, Griefensci, Switzerland) equipped with an in cooler and refrigerated cooling system was used to analyse the thermal behaviour of Levofloxacin and DRC. Sample (5-10 mg) was heated in hermetically sealed aluminium pans at temperature 20°C/min. nitrogen was purged at 50 ml/min and 100 ml/min through cooling unit.
Optimization and data validation
A total Thirteen batches of beads were prepared by choosing nine possible combinations while centre point was repeated four times and the mean value was taken into consideration for further study. The data obtained was analysed using Design Expert® 8.0.7.1 (trial version). The models were tested for significance. Validation study was conducted by developing four different batches of beads employing response surface methodology.
Evaluation of prepared beads
Characterization of beads
Several sharp peaks in XRD spectra of pure drug represent the crystalline nature of drug while a diffused peak in XRD spectra of represents amorphous nature of resin. But on the other hand XRD pattern of DRC shows disappearance of characteristic peaks of drug and also found to be broadened,these finding suggest the formation of drug resin complex.(fig 1)
Example 7:
In vitro drug release
The in vitro drug release of the formulated beads is shown in fomrulations were studied for their drug release profile for up to 8 h and it was observed that the rate of drug release may vary because of different concentrations of independent variables are 69.88 % to 82.13 %. (Table 2)
Table 2: Characterization of formulation beads
Batch Code Sodium Alginate
(mg) Chitosan
(mg) % Yield Particle
Size
(mm) Drug entrapment
(%) Drug release
(%)
F1 2 0.77 77±0.125 1.37±0.56 48.8±0.221 77.93±0.241
F2 4 0.71 80±0.121 1.26±0.751 54.3±0.231 73.91±0.222
F3 6 0.70 83±0.58 1.15±0.11 67.1±0.821 69.88±0.313
F4 2 0.45 74±0.721 1.33±0.222 35.1±0.221 80.03±0.562
F5 4 0.55 77±0.11 1.21±0.214 44.81±0.521 76.95±0.881

While the invention has been described with respect to specific composition which include presently preferred modes of carrying out the invention, those skilled in the art will appreciate that there are numerous variations and permutations of the above described embodiments that fall within the spirit and scope of the invention. It should be understood that the invention is not limited in its application to the details of construction and arrangements of the components set forth herein.
Variations and modifications of the foregoing are within the scope of the present invention. Accordingly, many variations of these embodiments are envisaged within the scope of the present invention.
The foregoing descriptions of specific embodiments of the present invention have been presented for purposes of description. They are not intended to be exhaustive or to limit the present invention to the precise forms disclosed, and obviously many modifications and variations are possible in light of the above teaching.
The embodiments were chosen and described in order to best explain the principles of the present invention and its practical application, and to thereby enable others skilled in the art to best utilize the present invention and various embodiments with various modifications as are suited to the particular use contemplated. It is understood that various omissions and substitutions of equivalents are contemplated as circumstances may suggest or render expedient, but such omissions and substitutions are intended to cover the application or implementation without departing from the spirit or scope of the present invention.
, C , Claims:1. A method for preparing controlled released chitosan-sodium alginate beads containing Levofloxacin Hydrochloride, comprising the steps of:
(a) preparing a sodium alginate solution by dissolving 2%, 4%, or 6% (w/v) sodium alginate in distilled water to form a clear solution,
(b) adding Levofloxacin Hydrochloride to the sodium alginate solution while stirring,
(c) preparing a chitosan solution by dissolving 0.25%, 0.50%, or 0.75% (w/v) chitosan in a 5% (v/v) acetic acid solution,
(d) dissolving calcium chloride in the chitosan solution to achieve a final concentration of 3% (w/v),
(e) adding the Levofloxacin-containing sodium alginate solution dropwise into the chitosan-calcium chloride solution using a 21-gauge needle to form microbeads,
(f) allowing the formed beads to cure for 30 minutes,
(g) collecting the beads by filtration,
(h) washing the beads with water, and
(i) drying the beads until a constant weight is achieved.

2. The method of claim 1, wherein the sodium alginate concentration is selected from the group consisting of 2%, 4%, and 6% (w/v), and the chitosan concentration is selected from the group consisting of 0.25%, 0.50%, and 0.75% (w/v).

3. The method of claim 1, wherein the Levofloxacin Hydrochloride is added to the sodium alginate solution in an amount effective to provide controlled release of the drug from the beads.
4. The method of claim 1, wherein the curing of the beads is performed at room temperature for 30 minutes.

5. The chitosan-sodium alginate microbeads produced by the method of claim 1, wherein the beads are suitable for controlled drug delivery of Levofloxacin Hydrochloride, exhibiting a prolonged release profile.

6. The chitosan-sodium alginate microbeads produced by the method of claim 1, wherein the beads are biologically compatible, nontoxic, and has excellent antibacterial qualities in order to increase its antimicrobial activity and decrease cytotoxicity.

7. The chitosan-sodium alginate microbeads produced by the method of claim 1, wherein the drug release is 69.88 % to 82.13 %.

8. The chitosan-sodium alginate microbeads produced by the method of claim 1, wherein the % yield is 74.07% to 83.58%.

9. The chitosan-sodium alginate microbeads produced by the method of claim 1, wherein the sarticle size is 1.15mm to 1.37mm.

10. The chitosan-sodium alginate microbeads produced by the method of claim 1, wherein the drug entrapment (%) is 35 to 67.

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