Introduction:
Currently, much interest is focused on the encapsulation of biologically active proteins in sustained released devices. Especially microparticles composed of poly (lactic-co-glycolic acid)(PLGA) extensively[1]. In biomedical field various formulations are based in microparticles, aiming the masking of unpleasant tastes and odors, controlled release of drugs, protection of drugs from aggressive body fluids, such as gastric juice, isolation of cells and development of immunoassays [2-3].
Increasing numbers of therapeutic agents emerging from rapid growth of biotechnological research components of pharmaceutical drug discovery are macromolecules such as proteins [4-6]. The advances in recombinant DNA technology have ensured the easy and abundant availability of several genetically engineered regulatory and therapeutic proteins such as hormones, growth factors, vaccines, immunosuppressors and so on. However, their unstable and acid labile nature, short biological half life, low bioavailability via certain routes large molecular size etc have limited effective role [4,7]. Biotechnology based products, especially protein drugs like hormones and their analogues, growth factors, vaccines etc are therapeutically active only by the parenteral route, and do not lend themselves administration by the oral route because of protein inactivation by acid and proteases of gastrointestinal tract [8-9].Different kinds of microparticles are usually manufactured by using polymers as the shell or matrix materials where those biodegradable and biocompatible are the polymers of choice [10]. These systems further present the versatility of allowing the incorporation of suitable amounts of drugs and improving the bioavailability of degradable drugs [11]. Microparticles also show the advantages of providing a large surface area, easier estimation of mass transfer behavior and precise kinetics modeling and controlled release of drugs to the body fluid [12].Poly(D,L-lactide-co-glycolide) (PLGA) is a biocompatible, bioabsorbable, and biodegradable polymer having low immunogenicity and toxicity ,approved by US Food and Drug Administration (US FDA) that is used to formulate many types of implantable and injectable drug delivery systems for humans and other animals. [13-15]. PLGA is synthesized by ring-opening of lactic acid and glycolic acid monomers and linkage via ester bonds. The molar ratio and sequential arrangement of lactide-glycolide units determine the physicochemical properties of the copolymer. The great success of 
PLGA-based microparticles as controlled drug delivery systems can be attributed to the various advantages this type of dosage forms offer, including: (i) the possibility to control the release rate during periods, which can range from a few days up to several months, (ii) relatively easy administration using standard needles and syringes (compared to the surgical insertion of implants), (iii) complete biodegradability, and (iv) good biocompatibility, even with brain tissue [16]. Lysozyme is selected as a “model protein” for encapsulation into PLGA using a double emulsion solvent extraction / evaporation method. However, it is often overlooked that lysozyme has an unusually stable three dimensional conformation and high isoelectric point of pH [17-18].
PLGA-based microparticles as controlled drug delivery systems can be attributed to the various advantages this type of dosage forms offer, including: (i) the possibility to control the release rate during periods, which can range from a few days up to several months, (ii) relatively easy administration using standard needles and syringes (compared to the surgical insertion of implants), (iii) complete biodegradability, and (iv) good biocompatibility, even with brain tissue [16]. Lysozyme is selected as a “model protein” for encapsulation into PLGA using a double emulsion solvent extraction / evaporation method. However, it is often overlooked that lysozyme has an unusually stable three dimensional conformation and high isoelectric point of pH [17-18].2. Materials and methods:
Materials:
Poly(DL-lactide-co-glycolide (PLGA ,85:15, Mw 50,000-75,000), lysozyme (from chicken egg white) and Micrococcus lysodeikticus were procured from Sigma-Aldrich Chemicals Pvt. Ltd., Bangalore, Karnataka, India. Polyvinyl alcohol (PVA, M.W. 30,000-70,000) was obtained from S.D. Fine Chem. Ltd., Mumbai, Maharastra, India. Dichloromethane (DCM) was purchased from E. Merck Ltd., Mumbai, Maharastra, India. All other chemicals used were of analytical grade.
Method of preparation of microparticles:
Required amount of PLGA (85:15) (Table 1) was taken in a beaker and dissolved in 20ml of dichloromethane (DCM). Then, the above solution was added to 2.5% of PVA solution containing lysozyme and was homogenized for 5 min at specified rpm. This primary w/o emulsion was added drop wise to 75 mL of 1.5% PVA solution in a 500 mL glass beaker and homogenized for 6 min at specified rpm (Table 1) to produce w/o/w emulsion. The emulsion was kept in rota vapour under vacuum to remove organic solvent for 15 min. Then it was stirred using magnetic stirrer overnight to evaporate remaining organic solvent. The microparticles were then washed thrice using double distilled water by centrifugation at 4,000 rpm for 20 min. Then the separated microparticles were freeze dried at 0.2 mbar, -400C, for 24 h using lyophilizer and stored the Samples.
| Formulation code | Stirring speed (rpm) | lysozyme :PLGA ratio |
| A1 | 4,000 | 1:40 |
| A2 | 5,000 | 1:40 |
| A3 | 6,000 | 1:40 |
| A4 | 7,500 | 1:60 |
Table 1: Batch specification
Optimization of protein-loaded microparticles:
Fourier transform infrared (FTIR) spectroscopy:
Lysozyme, mixture of PLGA with PVA and mixture of lysozyme with PLGA and PVA were mixed separately with IR grade KBr and were pressed to disk. Infrared (IR) spectra of the samples were scanned in the range from 400 to 4000cm−1 and recorded on a Fourier transform infrared spectrometer (Nicolet, Madison, WI, USA) to study the possible interactions between lysozyme protein and excipients.
Particle size and morphology:
The surface morphology and size of the microspheres were assessed by a scanning electron microscope (SEM), (JEOL, JSM5200, TOKYO, Japan). The powder sample was spread on a SEM stub and gold coating was done by using an ion-sputtering device. The gold-coated samples were vacuum dried and then examined. Particle size was obtained by measuring the diameters of at least 300 particles shown in SEM using image analysis software (Image-Pro Plus 4.5; Media Cybernetics, Silver Spring, USA). The size of each sample was measured at triplicate and the mean value was calculated.
Estimation of polydispersibility index:
Polydispersibility was performed by the instrument Zetasizer nano ZS(0.6nm to 6 µm) with DTS software (Malvern Instrument Limited, UK). NIBS® (non invasive backscatter optics) technology which improves sensitivity was used for measurements of particles. The formulations were taken in lyophilized form in microcentrifuge tubes, suspended in phosphate buffer, pH 7.4 and introduced in the instrument to read the results.
Estimation of zeta potential:
Zeta potential of different formulations was measured by the instrument zetasizer nano ZS using DTS software (Malvern Instrument Limited, UK) using M3-PALS (phase analysis light scattering) technology. The experimental formulations were taken in lyophilized form in 2 mL eppendroff tube and the samples were suspended in phosphate buffer, pH 7.4 and then introduced in the instrument following the guideline of the manufacturer. The results were then read.
In- vitro release study:
The PLGA microspheres, 5mg of each formulation, were weighed and individually suspended in 2ml phosphate buffer pH7.4 in test tubes and incubated in a shaker (Grant instruments, Cambridge, England) at 37 °C with constant shaking. The samples were kept for specified periods of time (5, 10, 15, 20, and up to 50 days, respectively) and were analyzed for protein determination. Fresh replacement media was added to resuspend the microspheres. The in vitro release experiments were conducted in triplicate. The cumulative amount of insulin released was calculated using the following equation:
Cumulative amount of lysozyme released (%) = Mt/ M∞× 100
Where, Mt is amount of lysozyme released at time t and M∞ is the total amount of lysozyme released at time infinity, which is the actual loading of lysozyme determined in loading efficiency experiment.
Protein-loading study
Lysozyme loading was determined by dissolving an appropriate amount of microspheres in a solution of 5% SLS-NaOH. After a definite period of time, the dispersed phase was separated from the continuous phase by means of centrifugation. Then the supernatant was collected and released protein was assayed spectrophotometrically using Lowry Protein assay Kit.
Results and discussion:
FTIR spectral analysis:
Drug-excipient interaction study is an important study to formulate a chemical entity with the other chemicals in a sustained release delivery system, so that the desire release pattern and other requisite physicochemical characteristics may be achieved (Mukherjee et al 2005a). Among the various methods available, FTIR spectroscopy provides us a distinct idea regarding interaction between various functional groups present in drug and excipients (Cunha-Filho et al 2007).
The level of interaction between lysozyme and PLGA at the level of functional groups through FTIR spectroscopy has not been reported so far in the literature. In the present study when the FTIR spectrum of lysozyme, PLGA-PVA and lysozyme with PLGA-PVA (Figures 1, 2, and 3) were compared, no physico-chemical interaction was detected. . Hence, PLGA and PVA were selected to formulate lysozyme loaded microparticles.
TABLE 2: Optimization of protein-loaded microparticles
| Formulation code | Average size | Polydispersibility index | Zetapotential (mv) | Mobility | Conductivity (ms/cm) | Loading capacity(%) ±SD (n=3) |
| A1 | 100 | 0.045 | -8.645 | -1.896 | 7.67 | 21.30±0.045 |
| A2 | 84 | 0.038 | -9.174 | -1.195 | 8.29 | 26.43±0.029 |
| A3 | 43 | 0.027 | -9.738 | -1.195 | 10.21 | 29.57±0.018 |
| A4 | 15 | 0.016 | -8.926 | -0.674 | 11.83 | 35.19±0.034 |
Surface morphology of lysozyme-loaded PLGA microspheres:
Particle size and size distribution are two important criteria of microspheres as these factors affect drug release rate, biodistribution, mucoadhesion, cellular uptake. The microspheres with a narrow size distribution are necessary to optimize the clinical outcomes. SEM photograph (Figure 1) shows that the surface-modified PLGA microspheres prepared by the double emulsion method were spherical in shape with smooth surface. The surface of these microspheres as observed under scanning electron microscope (SEM) was free from any pores or cracks (Figure 1). The particle size of individual microsphere was in the range of 15 μm to 100 μm (Table 1) and a major portion of the particle distribution occupied by very small particles in micro range size as compared to the larger particles which were also below 100 μm in size. This was confirmed by particle size analysis. There was a clear distribution of both the small and large particles and they were not conglomerated.
The average particle sizes of the formulations A1, A2, A3 and A4 prepared at 4,000, 5,000, 6,000, and 7,500 rpm respectively. The data (from Table 2) suggest that with increasing homogenizing speed, the average diameters of particles were reduced. However, when speed of homogenization was further enhanced (at 10,000 rpm), it was found interestingly that the average diameters of the particles were increased again, and became in larger micron range. Higher energy due to higher homogenizing speed might have agglomerated microspheres immediately after formation to provide larger spherical structures due to variable surface charges or any other similar cohesive forces generated due to the high energy process.
The results of particle size analysis by laser diffraction showed that particle sizes varied from 15 μm to 100 μm with variable polydispersity indices among the different experimental formulations (Table 2). Table 2 shows the various process parameters of the prepared protein containing microparticles. Formulation A4 had the least value of the average diameter, whereas A1 had the maximum. The polydispersibility indices also showed the similar patterns of dispersibilities i.e, formulation A4 had the least value and A1 had the maximum.
Figure 1 SEM photograph of the formulation A4.
Electrostatic characteristics of lysozyme-loaded PLGA microspheres:
The zeta potential is increasingly being used to investigate fine particle systems and representative of particle charge. The magnitude of the zeta potential gives an indication of the potential stability of the system. The findings of zeta potential study are given in Table 2. If all the particles have a large negative or positive zeta potential they will repel each other and there is dispersion stability. If the particles have low zeta potential values then there is no force to prevent the particles coming together and there is dispersion instability. A dividing line between stable and unstable aqueous dispersions is generally taken at either +30 or -30mV. Particles with zeta potentials more positive than +30 mV and more negative than −30 mV are normally considered stable for colloidal dispersion. From the data it is evident that all the formulations are unstable in the colloidal state. This suggests that the particles should not be stored in a liquid suspension form and rather they should be stored in a lyophilized state. Immediately before the administration they should be reconstituted. Reports suggest that formulations stored in colloidal stage cause more stability problem than in the dry form (19). All the formulations had negative zeta potential values varied between −8.645 to −9.738 mV. A4 had the value of –8.926 mV whereas A1 had −8.645 mV. When mobility was compared A4 had a lower mobility value of -0.674 as compared to that of A1, which was -1.896. Conductivity was found to be the highest (11.83ms/cm) in A4 among all the experimental formulations and the least value was seen in A1, which was 7.67 ms/cm.
In- vitro release study:
The release profiles of lysozyme from all formulations are reported in Fig. 5a and b. The incomplete release may be due to the fact that microparticles had not yet entered the degradation phase in which polymeric microparticles degrades and releases its remaining drug content, or to incomplete detachment of the protein adsorbed on the particle surface [20].
In addition, a major mechanism for release of many drugs is diffusion through water-filled pores formed by erosion. The pores are formed as polymer degrades and generates monomers and oligomers which are small enough to be soluble in the surrounding medium and to diffuse out of the particles. These small products are formed more quickly following degradation of lower molecular weight polymers; in other words, fewer bonds must be hydrolyzed to generate soluble oligomers as initial molecular weight decreases [21,26]. This will lead to higher release rate of protein. Cui et al. showed that there was a correlation between insulin release and the molecular weight of PLGA. An increase in the molecular weight of PLGA led to a reduction in both the initial and final release over 24 h. This could be explained by the retardant effect of the polymer meshwork generated by the longer chains in the PLGA with higher molecular weight [34].
In agreement with our results, polymer concentration showed to be a key factor to influence the characteristics and release profiles of microspheres [25]. It was reported
that the solidification of microspheres is more rapid at a high PLGA concentration, which may result in a viscous polymer layer at the microsphere droplet. features satisfying the objective of the study.
The average (n = 3) protein loading in A4 formulations was found to be 35.19% w/w of the formulations. Protein release study was carried out to understand the protein release pattern from the formulations. After 10 days release of the protein was about 30% of the protein content. When protein release study was conducted it was found that the cumulative amount of the protein release in 50 days was 83.52 μg per mg of formulation in case of G1. The values were 82.31, 72.35, 60.75 μg per mg of formulation in case of G2, G3 and G4, respectively (Figure 6). When the cumulative percentages of the protein release from the experimental formulations were plotted against time, it was found that about 84% protein was released in 50 days in case of the formulation G3, whereas, G4 had a release of about 42% in the same time period (Figure 7). G1 and G2 had a cumulative percentage release of about 61% and 77%, respectively. From the release kinetics it was evident that formulation G1, G2 and G3 followed zero order kinetics, whereas, G4 was closest to apparent zero order kinetic (Table 3).Protein loading is found to vary due to formulation compositions, methodology, process parameters etc. PLGA (50:50) was reported to have protein loading of 8-16% (Lgartua et al 1998), which is about 2-4 times more ie, ∼38% in the experimental formulations prepared with PLGA (85:15), in the present study. Improved BSA loading was achieved in the prepared formulation G4 (38.6%) in the present study. The protein-loading studies show that with increase in speed of homogenization, the loading capacity of formulation was increased. Likewise, the increase in amount of polymer also enhanced the loading of protein. High homogenizing speed might vary the surface charges, which ultimately could load more protein (Basarkar et al
2007). The in-vitro protein release study was carried out for a period of 50 days. We studied a pretty long period for studying sustained release of protein. However, literature showed variable protein release study period from 35 days to 70 days (Zhu and Schwendeman 1999; Leach et al 2005).
2007). The in-vitro protein release study was carried out for a period of 50 days. We studied a pretty long period for studying sustained release of protein. However, literature showed variable protein release study period from 35 days to 70 days (Zhu and Schwendeman 1999; Leach et al 2005).
Poly (lactide-co-glycolide) (PLGA), aliphatic polyester, has been well documented for its excellent biodegradability, biocompatibility and nontoxic properties (Loo et al 2004). PLGA varieties differ in their lactide-glycolide ratios which make PLGA varieties available in different molecular weights with variable physicochemical properties .
The mechanical strength, swelling behavior, capacity to undergo hydrolysis, and subsequently the biodegradation rate are directly influenced with the lactide content of the PLGA polymer (Jain 2000). Present study was conducted with a more uncommon PLGA (85:15) than the commonly used PLGA (50:50) variety. Lactic acid is more hydrophobic
than glycolic acid and hence lactide-rich PLGA (85:15) is less hydrophilic, absorbs less water, and subsequently degrade more slowly than PLGA (50:50) (Jain 2000). PLGA polymer containing lactic and glycolic acids in 85:15 ratio is hydrolyzed much slower than the PLGA (50:50) containing equal proportion of lacdide and glycolide. Molecular weight is indicative of chain length of polymers. By varying PLGA molecular weight, degradation of the polymer and the release kinetics of BSA can be controlled. Higher molecular weight of PLGA (85:15) than PLGA (50:50) due to the presence of higher lactide chain lengths enhances the hydrophobicity and decreases the degradation of the polymers and rate of protein release. In the present study, the nanoparticles containing larger particles with the increased
The mechanical strength, swelling behavior, capacity to undergo hydrolysis, and subsequently the biodegradation rate are directly influenced with the lactide content of the PLGA polymer (Jain 2000). Present study was conducted with a more uncommon PLGA (85:15) than the commonly used PLGA (50:50) variety. Lactic acid is more hydrophobic
than glycolic acid and hence lactide-rich PLGA (85:15) is less hydrophilic, absorbs less water, and subsequently degrade more slowly than PLGA (50:50) (Jain 2000). PLGA polymer containing lactic and glycolic acids in 85:15 ratio is hydrolyzed much slower than the PLGA (50:50) containing equal proportion of lacdide and glycolide. Molecular weight is indicative of chain length of polymers. By varying PLGA molecular weight, degradation of the polymer and the release kinetics of BSA can be controlled. Higher molecular weight of PLGA (85:15) than PLGA (50:50) due to the presence of higher lactide chain lengths enhances the hydrophobicity and decreases the degradation of the polymers and rate of protein release. In the present study, the nanoparticles containing larger particles with the increased
polydispersity indices showed slower release of BSA and it might be due to longer diffusion pathways of BSA in larger particles. However, more content of PLGA (85:15) showed the slowest BSA release in G4 among the formulations studied and it might be due to the enhancement of hydrophobicity. In the present study, the protein loading shows that increase in speed of homogenization increased the loading capacity of the formulation. Likewise, the increase in amount of polymer also enhanced the loading of protein. Increased entrapment efficiency was observed for particles of PLGA (85:15) with than PLGA (50:50), for their higher molecular weight and stronger hydrophobicity (Mittal et al 2007). Higher homogenizing speed might vary the surface charges of the polymers (Basarkar et al 2007), which ultimately could load more protein. In a study nanoparticles based on PLGA (85:15) without drug was showed to produce less polydispersity index than those with PLGA (50:50) (Mittal et al 2007). About 5% statisti- cally nonsignificant enhancement of nanoparticle sizes with PLGA (85:15) than PLGA (50:50) was reported in the literature (Mittal et al 2007). This indicates that increase lactide chain may not have great influence on particle size; rather the methodology has direct impact on the size reduction. In our study, variable homogenizing speed was found to be responsible for the size of nanoparticles and their polydispersity index.
Figure 2 and Figure 3 summarize the findings of in-vitro protein release study. Particles with smaller average diameter showed slower release. Smaller particles are generally formed with higher impact (Chen et al 2007). It varies the tortuous polymeric diffusion pathways in smaller particles (Zhang et al 2004). This ultimately leads to a sustained diffusion of protein from the particles. Formulations prepared with a lysozyme: PLGA ratio of 1:60 showed the slowest release pattern, among the formulations studied. Thus incorporation of extra amount of hydrophobic polymer, PLGA, might have caused more tortuous polymeric networks to deliver the protein for prolonged period of time maximally, among the experimental formulations. The method provided a narrow-ranged densely-dispersed microparticles having capability of release of protein (lysozyme) more than 50 days.
Thus, by regulating different process parameters and using a very simple method, BSA-loaded nanoparticles with a low polydispersity index can be produced and improved BSA loading and prolonged release profiles of BSA can be achieved. : degrade more slowly than PLGA (50:50) (Jain 2000). PLGA polymer containing lactic and glycolic acids in 85:15 ratio is hydrolyzed much slower than the PLGA (50:50) containing equal proportion of lacdide and glycolide. Molecular weight is indicative of chain length of polymers. By varying PLGA molecular weight, degradation of the polymer and the release kinetics of BSA can be controlled. Higher molecular weight of PLGA (85:15) than PLGA
(50:50) due to the presence of higher lactide chain lengths enhances the hydrophobicity and decreases the degradation of the polymers and rate of protein release
(50:50) due to the presence of higher lactide chain lengths enhances the hydrophobicity and decreases the degradation of the polymers and rate of protein release
Figure 2 cumulative amount of Lysozyme released from the experimental formulations.Data shows mean ± SD (n=3)
Figure 3 cumulative percentage of lysozyme released from the experimental formulations. Data shows mean ± SD (n=3) Table 3 : Release kinetics of lysozyme
| Formulation code | Zero order kinetics | First order kinetics | Higuchi kinetics | Kors-meyer-peppas |
| A1 | y = 0.388x + 9.250 R² = 0.990 | y = 0.008x + 1.362 R² = 0.977 | y = 7.738x + 3.772 R² = 0.960 | y = 0.405x + 1.059 R² = 0.936 , n=0.405 |
| A2 | y = 0.350x + 10.91 R² = 0.986 | y = 0.007x + 1.507 R² = 0.983 | y = 8.650x + 11.03 R² = 0.957 | y = 0.349x + 1.246 R² = 0.937, n=0.349 |
| A3 | y = 0.304x + 13.52 R² = 0.991 | y = 0.006x + 1.629 R² = 0.981 | y = 8.455x + 22.46 R² = 0.973 | y = 0.283x + 1.417 R² = 0.935 , n=0.283 |
| A4 | y = 0.094x + 7.908 R² = 0.973 | y = 0.004x + 1.453 R² = 0.957 | y = 3.150x + 21.22 R² = 0.971 | y = 0.185x + 1.313 R² = 0.957, n=0.185 |
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