PCL Quercetin Microspheres With Biopolymeric Surface Injectable Implants Biology Essay

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Collagen coated PCL-Quercetin microspheres was formulated for trafficking microparticle in biological environment and it act as a perfect biomaterials due to superior biocompatibility, low immunogenicity, low antigenicity. PCL-Quercetin microspheres were prepared by solvent evaporation technique using gelatin (0.5%) as a stabilizer but the burst release and immunogenicity is the major issues. It could be solved by collagen coated with PCL microspheres as an injectable implants. Native collagen (Type I) is isolated from rat tail tendon and estimated by the Woessner method. Freshly isolated collagen was coated with optimized PCL microspheres with different concentration to ensure that coating by FTIR and particle size analysis. The higher concentration collagen (3mg/ml) is well coated with microspheres. The encapsulation efficiency and in vitro release profile of collagen coated PCL-Quercetin were determined by UV spectrophotometer. Surface morphology of optimized formulations was characterized by optical microscope (OM) and scanning electron microscope (SEM). Drug and polymer interactions were studied by X-ray diffraction (XRD) and differential scanning calorimetriy (DSC). The studies are reported that the collagen coated microspheres have well defined release rate, surface morphology and also control the burst effect.

Key words: Collagen, Quercetin, Polycaprolactone, Injectable implants


Microencapsulation has an excellent advantageous novel drug delivery system as compare to conventional dosage forms; consequently it protects the active components, decreasing dose frequency, controlled drug release, taste masking effect and avoids the systemic side effects. Solvent evaporation method is very simple but effective method to produce spherical microparticles at constant stirring in optimized temperature1.

A numerous variety of synthetic or natural polymers have been employed as the implantable biomaterials and controlled-release drug delivery system in humans2. The synthetic or natural polymers like collagen, gelatin, and alginate, poly (dl-lactide-co-glycolide) (PLG) and poly (-caprolactone) (PCL) have been employed as the controlled release drug delivery system and development of implantable biomaterials3.

PCL is semi crystalline polyester, has an increasing attention as a material for controlled release formulations in the injectable implants. PCL and its derivatives, with a high permeability to many therapeutic drugs, lack of toxicity are well suited for drug delivery4 and also it does not produce the acidic environment while degradation like polylactide (PLA), polyglycolide (PGA) which cause degrade the active component5. PCL microsphere shows very severe burst release than the P (LA-b-CL) microspheres and PLA microspheres at the first day6. To control the burst release, mimic the natural body substance and improve the biocompatibility of the formulation by coating with natural polymers.

Collagen has increasing attention rather than the other natural polymers. Because it has low immunogenicity, low antigenicity, high biocompatibility and also collagen is the major components of extracellular matrix. It supports cell growth on the particle surface thus degrade the microparticles 7, 8 and enhance the implant-tissue interface 9 action. Most of the result the collagen gives a positive result when it is given as an injectable10. There are several types of collagen was existed in research, among them type I is most abundant of the human body. It is found in tendons and the organic matrix of the bone11.

Quercetin (QU) - 3,3',4',5'-7-pentahydroxy flavone (Fig. 1) is a flavanoid composed of two benzene rings linked through a heterocyclic pyrone ring. The experimental data's shows that it possesses numerous beneficial effects on human health and act against various life threatening diseases12. At present many studies found that the oral absorption of QU in both humans and rats is low, variable and become insufficient bioavailablity, perhaps the degradation of QU in intestinal microflora particularly in the intestinal bacteria13. Overcome this problem and improve therapeutic efficacy to prepare biodegradable microspheres for injectable implant.

The goal of the research is to develop an effective injectable implant by collagen coated PCL-Quercetin microspheres for improve the biocompatibility, controlled release and avoid the burst release from the PCL-Quercetin matrix.



Quercetin (QU), Polycaprolactone (PCL) and Gelatin was purchased from Sigma-Aldrich Chemical Co., (USA). Dimethylsulphoxide (DMSO), Dichloromethane (DCM), Disodium hydrogen phosphate, Disodium hydrogen orthophosphate, Sodium hydroxide, Acetone, Ethanol were purchased from Merk ltd., (India) and Collagen was isolated from rat tail denton and it is estimated by Woessner method.


Preparation of PCL-Quercetin microspheres

PCL-Quercetin microspheres were prepared by solvent evaporation method 14 (Fig. 2). Gelatin is the only aqueous phase tried in these formulations. This organic phase (solvent containing drug and polymer) was slowly added drop wise into 500 ml of aqueous phase at various concentrations (0.25%, 0.50%, 0.75%, and 1%) under the mechanical stirring until evaporation of organic solvent. The microparticles were then recovered by filtration15, 16. Formulation conditions are given in (Table 1).

Particle size determination and polydispersity intex

Particle size were determined for all formulations by laser diffractometry (Microtrac) using the software Microtrac FLEX. Prior to measurement, samples were dispersed in 5mL aqueous solution and ultrasonicated for 30 sec. Poly dispersity index was calculated by following formula,

Polydispersity index =

Where, D 0.9 -90% D 0.5 -50% and D 0.1 -10% of particles from the particle size determination data. Higher polydispersiblity index values point out the high level of non-uniformity of the microparticles17.

Drug loading and entrapment efficiency

Weighed microspheres are dissolved in 200 ml of DCM in 2 ml eppendrof tubes and vortexes for 10 min to separate the drug from the polymer coating add 1800 ml of ethanol and the polymer was precipitated. The resulting solution was centrifuged for 10 min at 3000rpm. The 200 ml of supernatant was diluted with 3ml ethanol the absorbance measured by UV spectophotometry 14, 18.

Selection of formulation

According to the particle size and entrapment efficiency results suitable formulations are selected from the eight (A1 to H1) formulations for further coating with collagen. B1 and E1 are two best formulations with high encapsulation efficiency and optimized particle size. These selected microspheres are coating with collagen for further studies.

Collagen coating

Selected microspheres (B1, E1) are coated with collagen I which is freshly prepared. After drying PCL-Quercetin microspheres was taken in the 15ml eppendrof tube, then 5 ml of collagen19 was added and stored in a room temperature for 1 h (Table 2). The sample was centrifuged at 6000 rpm for 10 min to decant the supernatant and settled microparticles are washed multiple times with distilled water to separate the coated particle. The particles were again dried in room temperature and stored.

In vitro release studies

QU release from the optimized formulations (B1, B1C, E1 and E1C) were determined using phosphate buffered saline (PBS) pH 7.4 as the release medium at 37±1°C. Microspheres were suspended in 10 mL of the dissolution medium in a screw capped tubes (15 mL). The tubes were tumbled end-over-end at 30 rpm in a thermostatically controlled oven. At regular time intervals, the tubes were centrifuged at 1500 rpm for 10 min and the aliquots of the dissolution medium were withdrawn and the same volume of fresh PBS was replaced to maintain sink conditions. The dissolution medium was maintained at a constant volume by replacing fresh dissolution medium throughout the release studies. The concentration of QU in the release medium was measured by UV spectrophotometer at 370 nm14.


The selected formulations (B1, B1C, E1 and E1C) were analyzed through the optical microscope (OM) for confirmation of microspheres formation and shape of the particles.

Scanning electron microscopic (SEM) technique, the microspheres were sprinkled on to one side of adhesive stub. The stub was then coated with conductive gold with JEOL-JFC 1600 AUTO COATER and was examined under JEOL-JFC 6360 scanning electron microscope. The surface nature of PCL -Quercetin microspheres and collagen coating PCL Quercetin microspheres are determined.

Fourier transforms infrared spectral (FTIR) analysis

A Fourier transform infrared spectrometer was used to explore the functional groups present in the formulated microspheres. Selected samples were crushed with KBr to get the pellets. The spectra of QU, PCL, empty microspheres and QU-loaded microspheres B1, B1C (optimised) were recorded.

Differential scanning calorimetrical (DSC) analysis

Experimental samples were weighed (3mg) and placed into coated aluminum pans, after which water was added to give a final concentration of 30% (w/w) dry substance. The samples were left for 35 hr before heating in the DSC, and were scanned at a rate of 10°C/min with a temperature sweep starting at 20°C up to 400°C, under a nitrogen atmosphere. All the samples were analyzed in a TA Instruments (model Q100 MDSC).

Polymorphism characterization by powder X- ray diffraction analysis

The crystalline microstructure of collagen coated PCL microparticles is expected to have impact on behavior of drug release. X-ray patterns were obtained (SEIFERT model JSO-DEBYEFLEX-2002) with a cu Kα radiation, θ-2θ powder diffractometer set for an angle range of 5 -70° 2θ. The step size was 0.04° 2θ and count times were of 1 sec per step. The analysis was carried out on the Empty microspheres, B1, B1C formulations.


Preparation of Collagen coated PCL-Quercetin microspheres

The PCL has slow degradation rate and it is very advantageous for the formulation of a parentral controlled delivery system. The solvent evaporation method is the most common technique used for the preparation of PCL-Quercetin microspheres. Even if the method is simple but many factors play a role in produce effective PCL microparticles. The solvent evaporation method is easy to scale up, effectively produce microspheres. Evaporation of the DCM during the stirring the polymer droplets become particles with the help of gelatin containing aqueous phase.

Using PVA as a stabilizer the resultant microsparticles were irregular and broken shape. Nevertheless the gelatin as a, the microparticles possess smooth surface, smaller particle size as well as narrow particle size distribution 20. A mixture of DMSO (3ml) and DCM (7ml) is used as an organic phase to solubilize of QU and PCL. Stirring time is 2 h, which is found to produce the encapsulated microspheres by complete evaporation of DCM from the medium. The volume of the aqueous phase (500ml) is very important during the microspheres preparation. The PCL microspheres might be a triphasic in release profile due to the drug adsorbed on the surface of microspheres, a second release phase due to the drug diffusion through the pores or channels formed in the polymer matrix the third phase due to polymer biodegradable4. After preparation of PCL-Quercetin microspheres, it was coated with freshly prepared collagen. It is simple method of coating to the PCL-Quercetin microparticles. The Formulated microspheres were taken in eppendrof tube with various concentration of collagen, when it has lower concentration (1 mg/ml) the morphology of microparticles might be disturbed by acetic acid. But in higher concentration (3 mg/ml) the particles were very smooth and well coated. The phase one release profile (burst release) was controlled by the collagen coating over the microspheres.

In the present study PCL-Quercetin microspheres of QU are successfully prepared by using 500ml aqueous phase and it is coated with collagen. The influence of the polymer (PCL) concentration (5%w/v), collagen (3mg/ml), stabilizer (Gelatin) and effect the stabilizer concentrations (0.25%, 0.5%, 0.75% and 1%w/v) on particle size (PS), entrapment efficiency (EE) are investigated. Stabilizer concentration at 0.5% w/v produces a better entrapment efficiency (62.42%) and particle size (100.5µm).

Particle size determination and Poly dispersity index

Particle size is important factor which can influence the biopharmaceutical properties of the microspheres, product syringeability and in vivo fate by uptake of phagocytic cells21. The size of the QU microspheres prepared with collagen and PCL is increase compare to PCL alone preparations, it is concluded by the particle size analyzer reports (Microtrac FLEX) shown in Fig. 3. This result concluding that the coating material collagen is present in the loaded microspheres. Viscosity of the aqueous phase plays an important role in the optimized particle formulation. When increase the stabilizer concentration more than 0.5%w/v, the particle size was increased. The rotating speed of the stirrer is also the major factor for control the size. Particle size was decrease when increase with the rotation speed. The rotation speed of 1600 rpm was to produce an excellent and uniform microsphere with in 2 h. The PCL-Quercetin matrix microspheres (B1) has an average size of 100.5µm but when it is again coat with collagen (B1C) the particle size was subsequent increases 112.2µm, thus collagen was confirmed in that formulation.

Non-uniformity of microspheres was calculated by using poly dispersity index. The unequal size of the microspheres directly affects the drug release rate. Poly dispersity results are shown in (Table 3). From all formulation, B1 and B1C are seen to have narrow distribution of microspheres.

Drug loading and entrapment efficiency

The entrapment efficiency of microspheres prepared using gelatin as stabilizer is given in Table 3. The gelatin had shown similar trend on the influence of concentration on entrapment of QU i.e. increasing concentration of stabilizer from 0.50%w/v to 1%w/v shows decrease in entrapment efficiency of QU from 62.42% to 55.33% and 54.55%. Entrapment efficiency of the drug is increased with increasing concentration of polymer PCL. The polymer concentration was optimized (0.5% w/v) with that of aqueous phase concentration.

In vitro release

Based on the particle size analysis and entrapment efficiency formulations B1, B1C, E1 and E1C were selected for in vitro release studies. The QU release profile from collagen coated PCL - microspheres in phosphate buffer saline solution of pH 7.4 at 37°C, were found to be very similar for all four selected formulations. The percentage cumulative drug release of B1, B1C, E1 and E1C are represented as graphs shown in Fig 5. B1C and E1C formulation shows a better release than the B1 and E1. QU is either loosely associated with the surface in the surface layer are responsible for the burst release. Surface associated QU are widely known to be the main cause for the initial burst. PCL-Quercetin microspheres are having initial burst release. After the burst release the drug slowly released from amorphous region of PCL microspheres. The matrix microspheres burst release was controlled by coating with collagen. The formulation is an injectable implants so that the polymer should be mimic the natural body constituent might be avoid the immunogenic responses. Collagen coated that resulting microspheres had a decreased burst effect and controlled release.


The surface morphology of the microspheres prepared using gelatin resulted in lower particle size were characterized using SEM. The surface morphology of empty of microspheres, B1 and B1C formulations with same condition (0.5%w/v gelatin as a stabilizer) are shown (Fig 4). It is clearly seen from the OM and SEM that the selected microsphere formulations are spherical in shape and uniform size. The B1 formulations had shown some crystals on the surface. It might be reason for the drug present in the surface. B1C were of good morphological characteristics, spherical with smooth surface, there is no crystals on the surface and without any aggregation homogeneously compare to B1. It concluded that the collagen fully coated on the surface of the PCL-Quercetin microspheres.

Fourier Transform Infrared Spectral (FTIR) analysis

FTIR spectra obtained could be confirmed the functional groups present in the collagen coated PCL- Quercetin microspheres. Fig. 6 shows that FTIR spectra of QU, PCL microspheres without QU, Quercetin loaded PCL microspheres (B1), collagen coated PCL-Quercetin microspheres (B1C). QU shows characteristic peak around 1100 - 1600cm -1, -OH phenolic bending (1200 - 1400 cm-1) in free and encapsulated form of QU. The polymer, PCL, shows characteristic peak at 1730 cm-1 which corresponds to the C=O of ester carbonyl groups. The protein amide I band at 1600-1660 cm-1 (mainly C=O stretch) is associate with stretch vibration and amide II band at 1548 cm-1. The peaks at 2868 and 2947 cm-1 are related to the C-H bond of saturated carbons. These results indicated the presence of collagen in PCL microparticles. The FTIR spectrum of QU loaded PCL microspheres shows additional peaks due to QU in the blend matrix.

Differential scanning calorimetry (DSC)

Fig. 7 shows that DSC of PCL, QU, empty microspheres, PCL-Quercetin microspheres (B1), and collagen coated PCL-Quercetin microspheres (B1C). Melting temperature (Tm) or endotherm peaks of plain PCL, empty microspheres, and loaded microspheres, both PCL and collagen were observed at 59 °C. Melting temperature of QU was observed at 318°C and it shows broad endothermic peak for dehydration from 102 °C to 110°C. Low concentration of QU was dispersed in the matrix, that's raveled by QU peak was not prominent in the loaded formulation. These Collagen coated PCL microspheres did not contain any peaks associated with the crystals of the drug, suggesting that the drug is amorphous form in the PCL. It is also concluded that the QU is dispersed in the amorphous region of the polymer matrix not in the crystalline region and also that PCL maintained its semi crystalline characteristic in microparticles.

Powder X- ray diffraction (PXRD)

PXRD was performed on QU, empty microspheres, QU loaded PCL microspheres (B1), and collagen coated PCL-Quercetin microspheres (B1C) shown (Fig. 8). In the X-ray diffractogram of QU powder, sharp peaks at a diffraction angle of

2θ = 4.46 °, 11.64°, 26.26° and 27.18° are present which suggest that the drug is crystalline material. There are also two strong peaks in the diffractogram of plain PCL microspheres, QU loaded PCL microspheres and collagen coated PCL-Quercetin microspheres at a diffraction angle of 2θ = 21.17°, 23.42° shows its semi crystallinity nature, but the intensity of the QU loaded peak decreased indicating the change in semi crystallinity of the PCL. The absence of QU intensity peak showing its crystallinity in the QU loaded microspheres indicate that changed to amorphous form.


Freshly prepared collagen coated PCL-Quercetin had been shown to be an effective for controlled release of QU. However it is a great challenge to develop the collagen coated PCL-Quercetin microsphere as an injectable implants. The optimized formulation (B1C) shows controlled release of QU (50%) for more than 10 days. The QU loaded collagen-PCL microsphere prepared, because of collagen is an excellent tool for biomaterials, drug delivery system and it is appears to be an effective for controlling the burst effect and improving the release rate.