Preparation of antibacterial collagen–pectin particles for biotherapeutics

Abstract

Biomaterial microparticles have versatile applications in biomedical formulations in site specific targeting treatment due to their size. There are many types of particulate formulations available in the pharmaceutical industry. But still it is difficult to produce protein solid particles due to their high sensitivity to heat. In this study, collagen–pectin microparticles (solid aerosol) are prepared using an electrospray method under ambient temperature and pressure conditions in a single step process. Pectin is used as a stabilizing and natural antiglycation agent for diabetic wound treatment. Collagen molecules can form various structures and aggregation states in solutions. During microparticle preparation, the electrospray method might affect the particle morphology. The significance of particle morphology was investigated using various analytical techniques. The effect of acetic acid concentration was monitored by viscosity, conductivity, differential scanning calorimetry (DSC), X-ray diffraction (XRD) and Fourier transform infrared spectroscopic (FTIR) measurements. The surface modifications of the microparticles were studied using scanning electron microscopy (SEM) and atomic force microscopy (AFM). The optimized results indicate that the collagen–pectin microparticles are obtained by electrospraying collagen dissolved in 50–80% acetic acid with 0.25% NaCl. Collagen–pectin microparticles are obtained which measure less than 150 μm in size when sprayed using a 0.65 mm sized nozzle. The prepared microparticles were also tested for antimicrobial activity to ensure high efficiency use during wound infection. Thus, the collagen–pectin microparticles prepared can potentially be used as biomedical particles for clinical applications including wound healing and lung regeneration for diabetic patients.

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Introduction

Microparticles prepared from biodegradable polymers have been largely explored for various biomedical applications. Protein particles with controlled release help in various disease treatments like diabetes, hormonal dysfunction and immunity disorders.^1,2

Pectin is one of the most common biopolymers due to its advantages such as non-toxicity, processibility and biodegradability. Moreover, it has important role in plant growth, morphology, development, cell adhesion and plant defence and also works as an emulsifier, gelling and stabilizing agent in diverse food and specialty products. It also reveals defence mechanisms against plant pathogens and wounding. In the present study an approach is carried out to improve the cyto-compatibility of pectin by interacting with collagen through surface modification because collagen can specifically bind with integrins on cell membrane, which can effectively accelerate cell attachment and spreading.^3,4 Polysaccharide nanoparticles have acknowledged considerable attention over the years and playing a pivotal role in delivering cancer chemotherapeutics and genetic therapy drugs. It was anticipated that due to small volume, they can easily pass through tissue interstice and they can be absorbed by particular cells and can be removed by phagocytes. The properties of polysaccharide nanoparticles have to be altered depending on the specific requirements. Natural biodegradable materials have recently been investigated for preparation of nanoparticles because of their exceptional physiochemical properties and biocompatible nature which is beneficial for biomedical use. Recently thiolated pectin based nanoparticles has been developed and its potential for ocular drug delivery was investigated.

Electro-Spray Deposition (ESD) is a method to produce monodisperse droplets with varied size measurements.^5–9 In the present study this technique is employed to prepare the pectin based collagen microparticles.

Antibacterial effect of wound healing material is of great interest due to delay in curing process. Disclosure of subcutaneous tissue which results causes damage to structural integrity of skin provides environment that is conducive to microbial growth through a moist, warm, and nutritious conditions. Furthermore, the profusion and diversity of microorganisms in any wound will be prejudiced by factors such as type of wound and the antimicrobial efficacy of the host immune response.¹⁰ Pathogenic microflora are usually transferred by resources used for the treatment. The presence of infection chiefly depends on the type of micro-organisms present in the wound and the healing process depends on the micro-organisms strains and their pathogenicity. Generally, ulcerations contain diverse flora consisting of various bacterial strains. Aerobic microorganisms such as Escherichia coli, Streptococcus pyogenes, Staphylococcus aureus, Pseudomonas aeruginosa and other anaerobic microorganisms are also infect the wound areas.¹¹ So far, the mechanism by which bacteria hinder the healing process is not completely understood. It is postulated that the virulence factors of bacteria is primarily due to bacterial adhesions, proteins on the surface of bacterial cells which are responsible for their adhesion to host cells, which allows bacteria to grow, degradation of cell materials such as collagen and fibrogen. Release of toxins, protein substances is also an indicator of bacterial infection.¹² In the present study, collagen–pectin microparticles are evaluated for antimicrobial efficiency to overcome this clinical wound diseased conditions.

In the current investigation, we demonstrate the capabilities of the ESD technique for the preparation of biopolymer based microparticles containing protein. Pectin–collagen based microparticles is prepared using ESD for biotherapeutics. In our previous work¹³ collagen particles are prepared using ESD and used as a carrier in drug delivery systems. This study aims at preparation and characterisation of pectin based collagen microparticles with antibacterial activities.

Results and discussion

Preparation and characterization of salt assisted collagen pectin microparticles – liquid phase characterization

During ESD spraying process, the main aspect of phenomena are controlling of charged droplets. The ionic conductivity was considered to be the preliminary requirement for an electrolyte. Fig. 1 shows the conductivity of 10 mg mL⁻¹ collagen–pectin solution with NaCl as a function of acetic acid concentration (50–80%). By increasing the acetic acid concentration, the conductivity is decreased slightly from 1000–400 μS cm⁻¹.

Fig. 1 Effect of neutral salts in the conductivity measurements as a function of acetic acid concentration in collagen–pectin complex solution.

This is mainly due to the ability of positively charged ion such as sodium (Na⁺) as major contributors and negatively charged ions such as chloride (Cl⁻) as minor contributors to conductivity. The ionic composition of the experimental solution also found to be critical in the spraying process. The study is initially carried out by subjecting 0.25% NaCl. It is observed that at 50% acetic acid concentration the conductivity of the solution increased more intensely in the range 998 μS cm⁻¹. But it shows a gradual decrease as the acetic acid concentration increases and reaches about 418 μS cm⁻¹ at 80% acetic acid concentration. This marked variation is mainly due to the presence of more free ions in 0.25% NaCl added collagen solution with high conductivity.

The solution property based on conductivity measurements varied markedly based on its swelling nature due to presence of polyions in the acid solubilised collagen solution. They form the aggregates as a uniformly charged domain and an undrained particle. The counter ions bound inside the domains have no contribution to the conductivity. In case of 0.25% NaCl addition, it was observed that subjected salts do not alter the structure of collagen to greater extent. The data indicates that some salts are bound it to collagen and it is more accessible for electrospraying process. This is because the sodium ions combine more prominently with the suspended carboxyl groups in the acetic acid solubilized collagen solution. The collagen solution with 0.25% salt addition was chosen for further characterization to obtain solid collagen–pectin microparticles from our previous study.

The particle size measurement of collagen–pectin solution before electrospraying was analyzed using Dynamic light scattering analysis. DLS studies of the collagen–pectin showed the critical aggregation behaviour in salt solution. The concentrations of collagen–pectin complexes are the evidence for significant increase in particle size. From Fig. 2, it is evident that the particle size of collagen–pectin molecule was found to be relatively decreasing as a function of acetic acid solution. Before spraying, it had a narrow size particle distribution in the native collagen solution, with an average size of 3.5–1.2 μm as a function of acetic acid concentration. Moreover, the polydispersity of the solution also shows a similar trend in the presence of neutral salts as a function of acetic acid concentration.

Fig. 2 Particle size distribution and polydispersity of collagen–pectin microparticles at different acetic acid concentration in the absence and presence of NaCl.

The collagen–pectin molecule carries a net positive charge at acidic pH conditions. The neutral salts may account for drastic changes in the protein molecule¹³ and leads to huge variation in the particle preparation (Fig. 3).

Fig. 3 Mechanism of interaction of collagen with pectin and its effect on salt induced mechanism.

The zeta potential variations for collagen–pectin complexes prepared at different concentrations of acetic acid (50, 60, 70 and 80%) are given in Table 1. Pectin interacted collagen exhibits a tendency to shift in lower potential value as a function of acetic acid concentration, evidencing the partial covering of the collagen molecule by pectin. The result clearly indicates that the rate of mobility of interacted complex decreased as compared to control inferring the extent of binding. This explains that collagen may have a variable number of amino acids with charged side chains, resulting in different surface charge values. The free or liberated ions in the medium might decrease the double layer effect.¹⁴

Table 1 Zeta potential measurements of collagen–pectin complex

Sample	Zeta potential (mv)	Mobility (cm^2 V^−1 s^−1)	Conductivity (mS cm^−1)	Doppler shift (Hz)	Base frequency (Hz)
0	42.50	3.314	0.5960	25.86	115.1
CP50Na	1.80	1.407	2.5354	1.08	114.5
CP60Na	2.11	1.648	2.0941	1.27	114.9
CP70Na	0.92	2.301	1.5506	0.55	115.6
CP80Na	−4.05	−3.157	1.141	−2.45	114.9

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Solid phase characterization – physiochemical analysis

Native collagen also displays bands at 1654, 1554 and 1240 cm⁻¹ which is characteristic of the amide I, II and III bands.¹² The effect of neutral salts on collagen especially at low concentrations (0.25%) are verified. An FTIR spectrum is recorded as a function of acetic acid concentration (50–80%). As the concentration of acetic acid increases, Amide I, II and III are shifted to 1533, 1410 and 1074 cm⁻¹, respectively. The presence of unordered conformation referred as random coil exerted an absorption peak at 1522 cm⁻¹. This indicates that interaction mode of collagen molecule binds to NaCl by carboxyl oxygen atoms and amino nitrogen in the collagen molecule. Moreover, in the presence of NaCl, amide A absorption band gradually disappears at very high acetic acid concentration. This characteristics change in the absorption peak clearly indicates the participation of NaCl in structural transitions. In microparticles a shift from 3420 to 3413 cm⁻¹ is shown which attributes to –NH₂ and –OH group stretching vibration. Moreover, Amide A peak becomes wider indicating that hydrogen bonding is enhanced (Table 2).¹⁵

Table 2 Amide absorption bands of crosslinked collagen–pectin (1 : 1) (CP) microparticles [1 : 1] with 0.25% content of NaCl at varying acetic acid concentration (50–80%)

Sample	Amide A (cm^−1)	Amide I (cm^−1)	Amide II (cm^−1)	Amide III (cm^−1)
CP50Na	3420	1533	1410	1074
CP60Na	3413	1525	1410	1072
CP70Na	3404	1524	1410	1072
CP80Na	3320	1522	1410	1070

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The DSC heating curve for collagen–pectin complex in the presence of salt as a function of acetic acid concentration is shown in Fig. 4. The characteristic endothermic peaks in the figure are termed as denaturation temperature (T_d). The electrospraying process is performed at high acidic conditions and salts substituent. The matrices prepared are characterized for their thermal stability as there is a chance for denaturation of the collagen molecule.¹⁶ As illustrated in Fig. 4, the DSC scans show an endothermal peak with a maximum at 120 °C for collagen solution dissolved at 50% acetic acid concentration. From the thermograms it is observed that as the acetic acid concentration increases, T_d slightly shifts to the lower temperature. Even at high acetic acid concentration it is observed that matrices are thermally stable of about 75–80 °C. Moreover, during the subsequent cooling and second heating cycle other transitions could not be detected.

Fig. 4 DSC profiles for collagen–pectin microparticles with the effect of acetic acid (50–80%) and added neutral salts (NaCl).

X-ray diffraction is used to investigate the collagen fibril distribution and orientation in mineralized tissues. Fig. 5 shows the XRD spectra of collagen–pectin complex. There are two diffraction peaks at the diffraction angles (2θ) about 7.5° and 20.1°. The first one is sharp but the second one is wide, which are in accordance with the characteristic diffraction pattern of collagen.¹⁶ The first peak are related to the diameter of the triple helix collagen molecule and second one with the single left-hand helix chain. Typical amorphous broad peak shifted at around 22° as seen in Fig. 5. From that data it is observed that it exhibit their characteristic amorphous broad peak for pectin. Whereas in case of NaCl, in addition to broad amorphous peak around 22° they also posses the two sharp characteristic peak around 26 and 32° for NaCl as seen in Fig. 5.

Fig. 5 X-ray spectrum showed collagen–pectin microparticles prepared by varying acetic acid concentration with the addition NaCl.

Hence, it is observed that collagen–pectin cannot crystallize during electrospraying and predominantly give similar amorphous structure in microparticles.

The effect of acetic acid dependency on average particle size of collagen–pectin microparticles and its structural morphology is shown in Fig. 6a–d. The electrospraying of collagen–pectin solution by increasing the acetic concentration resulted in spinning fibres without any salt addition. The neutral salts are used as an antidote to produce individual particles for drug delivery system. Hence, salts such as NaCl was chosen, optimized from their solution properties and studied for their dependency in particle production. The structural transition of collagen molecule in the presence of salts makes it more feasible for producing the relatively monodisperse collagen and collagen–pectin microparticles by electrospraying. As seen in Fig. 6a–d, the collagen–pectin microparticles obtained by addition of NaCl yielded quasi-monodisperse particles with average diameter of approximately 120 μm at 50% acetic acid.

Fig. 6 SEM images and volume mean sized distribution of collagen electrosprayed using 0.65 mm sized nozzle with added NaCl by varying acetic acid concentration such as 50% (a), 60% (b), 70% (c) and 80% (d) respectively. Electrospray liquid flow rate and current are 0.2 mL h⁻¹ and −25 kV. Magnification – 50 μm.

Generally the concentration of acetic acid increases particle size also increases in turn by decreasing solution conductivity.¹² In the presence of salts the conductivity increased much higher and provides a reverse effect on the solution property. By this means the relatively small solid particles is produced at 50% acetic acid itself. The variation in the structural forms at higher concentration of acetic acid, is failed to produce individual particles. Moreover, the solutions could not be electrosprayed when acetic acid concentrations lower than 50%. The particles produced are predominantly of doughnut shaped (Fig. 6a). The increasing production rate from a single cone by at least one order of magnitude can be achieved by augmenting the liquid flow rate.

The AFM images shows the collagen–pectin microparticles surface morphology similar to that of the SEM images obtained (Fig. 7a and b).

Fig. 7 Topographic images by AFM representing collagen–pectin complex in phase imaging (A) and topographic imaging prepared from 50% acetic acid with addition of 0.25% of NaCl.

The present study has shown that changes in the phase image of collagen helix transition are well confirmed from the AFM images as seen in Fig. 7a. Moreover, AFM enables the direct visualization of the surface topography of collagen molecules at higher magnification (Fig. 7b). The formation of collagen–pectin microparticles in the form of sickle or doughnut shaped structures by means of salt assisted process is distinctively observed. Moreover, the data from AFM images indicates that collagen–pectin molecule in the acetic acid medium are in the long range ordering in the form of dispersed helical forms or random coils without denaturation. Thus the results of these analysis methods indicate visibly the structural modifications in collagen–pectin molecule in the presence of neutral salts.

Clinical significance of collagen–pectin microparticles

Biomass estimation-turbidity measurements. Collagen–pectin microparticles of about 100 μg mL⁻¹ are used for the turbidity measurements. Growth curves of control strain such as Bacillus subtilis, Psuedomonas aeruginosa, Escherichia coli, Streptococcus pyogenes and Lactobacillus acidophilus and the strain treated with collagen–pectin microparticles (NaCl dependent) in LB medium inoculated with 10⁷ colony forming unit (CFU) are studied and recorded in Fig. 8. With respect to gram positive organisms such as Bacillus subtilis and Streptococcus pyogenes, NaCl treated microparticles caused a significant increase in growth delay with the increasing concentration of acetic acid concentration. Hence these concentrations showed a considerable biocidal activity at increasing concentration of acetic acid concentration. Microparticles with highest acetic acid concentration (100 μg mL⁻¹) showed almost no growth when inoculated on to the Muller Hinton agar medium for up to 24 h representing a bactericidal effect at this concentration (Fig. 8). Direct interaction between collagen–pectin microparticles and cell surfaces of gram positive organisms affects cell membrane permeability and lead eventually to the inhibition of growth and cell death. In case of Streptococcus pyogenes, a collagen–pectin microparticle has the ability to induce cellular protein leakage. Leakage of protein is marked as an indicator of cell membrane damage by the collagen–pectin particles. It has been suggested that the cytoplasmic membrane is found to be the target of intended action.

Fig. 8 Antibacterial activity of collagen–pectin particles.

In case of Lactobacillus acidophilus (gram positive microorganisms) showed antibacterial activity in the reverse manner compared to other gram positive organisms. Here, this species has the ability to with stand the high acetic conditions and show a sustainable growth compared to the other species. Antibacterial activity decreased with increase in acetic acid concentration containing microparticles. Although, there is a noteworthy inhibitory action compared to the control. Collagen–pectin microparticles are responsible for the mechanism of antimicrobial activity on probiotic effect of Lactobacillus sp.

Collagen–pectin microparticles used characteristically have an acidic pH, which is low enough to inhibit pathogenic bacterial species. In case of gram negative organisms (Escherichia coli and Pseudomonas aeruginosa) collagen–pectin microparticles with highest concentration (100 μg mL⁻¹) showed almost no growth for up to 24 h representing a bactericidal effect at this concentration (Fig. 8). The bactericidal effect on Escherichia coli is mainly due to the disruption of cell membrane integrity by the collagen–pectin microparticles. In case of Pseudomonas aeruginosa, collagen–pectin microparticles results in the leakage of the cytoplasmic component and lead to the eventual death of the organism.

Antimicrobial activity-zone of inhibition

The antimicrobial susceptibility test is performed by disc diffusion method against different microorganisms i.e., Bacillus subtilis, Pseudomonas aeruginosa, Escherichia coli, Streptococcus pyogenes and Lactobacillus acidophilus (Fig. 9). The controls for all the microorganisms are carried out in the absence of collagen–pectin microparticles. Zone of inhibition resulted against all the five microorganisms as given in Fig. 9. The results presented in the table attribute that collagen–pectin microparticles showed antimicrobial property against pathogens. Here, this study explains the effect of microparticles on antimicrobial susceptibility is more significant. Highest zone of inhibition is observed for Bacillus subtilis and Escherichia coli measuring about 28 mm at 90% acetic acid solubilised collagen–pectin microparticles. The influence of acetic acid and the neutral influences the activity of collagen–pectin microparticles on bacterial cell mass.

Fig. 9 Zone of inhibition of collagen–pectin complex against bacterial strains.

The gram positive bacteria such as Bacillus subtilis, Streptococcus pyogenes, Lactobacillus thermophilus are found to be susceptible to the activity of collagen–pectin microparticles indicated by the zone of inhibition measuring more than 10 mm. Except for the Lactobacillus acidophilus, Bacillus subtilis and Streptococcus pyogenes follows a similar trend undergoing maximum cell death with maximum zone of inhibition (>24 mm) at 90% acetic acid concentration with NaCl. With respect to Lactobacillus, a reverse trend has been followed indicating maximum inhibition at 50% acetic acid concentration prepared from NaCl (24 mm) and decreased as the acetic acid concentration increases. This is mainly due to the tendency of collagen–pectin microparticles on Lactobacillus to show high resistance towards cell death at higher % of acetic acid concentration. The activity of gram negative bacteria on collagen–pectin microparticles showed a same trend compared to that of gram positive bacteria. Collagen–pectin microparticles (NaCl) demonstrated a maximum zone of clearance of about 24–28 mm at higher % acetic acid concentration. The zone of inhibition data for both gram positive and gram negative explains the significant biocidal activity of collagen–pectin microparticles. The antimicrobial activity of collagen–pectin microparticles can be explained by its capability to penetrate the outer layer of cells of microorganism.

Evaluation of fibroblast cell proliferation rate of collagen–pectin microparticles

Cell viability (%) of collagen–pectin microparticles after 24 h exposures using MTT assay is presented in Fig. 10. Each experimental data represents the average of a series of three different experiments. Cell viability is found to be significantly increasing for collagen–pectin microparticles for CP50 and CP60 when compared with the control (only cells without collagen–pectin). However, in the case of CP70 and CP80 there is decrease in cell viability. This is probably due to higher concentration of acetic acid. It is well known fact that, collagen is usually prepared in acetic acid medium and then neutralised to physiological pH for biological functions. The optical density values are in similar trend with the microscopic images, as shown in Fig. 11.

Fig. 10 MTT assay of collagen–pectin particles of varied acetic acid concentration.

Fig. 11 Cell image at different concentration of collagen–pectin particles. (A) Control, (B) 50% AA, (C) 60% AA, (D) 70% AA and (E) 80% AA (magnification ×20).

The cytotoxicity study is carried out to understand the biocompatibility of the prepared microparticles. From the current research, it can be postulated that CP50 (50% acetic acid) is a optimum concentration to prepare collagen–pectin microparticles.

Conclusions

The present study shows that collagen–pectin microparticles can be prepared with reproducible size, in a size range less than 500 nm, by an electrohydrodynamic forming. Addition of neutral salts such as NaCl provided the basic improvements in the preparation procedure. The acetic and salt concentration in the collagen solution is identified as crucial parameters for particle stability. The novelty of the present work relies in producing efficient solid microparticles of doughnut or sickle shaped from the acetic acid solubilised collagen. This is a single-step and relatively simple process that does not expose proteins to harmful organic solvents and thus can be exploited to prepare biodegradable microparticles as a carrier for drug release. It is found the structural transitions are favoured by the presence of NaCl concentration, without affecting the conformation of collagen. The main findings from the present investigation refer to the stabilizing role of salts in preparing collagen–pectin microparticles. The surface morphology study using AFM and SEM of the electro sprayed collagen indicates that NaCl at a concentration of about 0.25% can help in producing the particles. Surface morphology measurements through AFM together with scanning electron microscopy imaging proved to be a suitable tool in the evaluation of the size and shape of the these biodegradable solid microparticles. The results obtained from the present study provides useful information for the future studies aiming at development of potential carriers for wound treatment consisting of antibacterial and biocompatible collagen–pectin microparticles.

Materials and methods

Type I collagen was purchased from Sigma Aldrich. Acetic acid was purchased from Wako Pure Chemical, Osaka, Japan. Pectin is purchased from Sigma. All chemicals were used without further purification. Ultrapure water was prepared using a water purification system (Milli-Q Biocel A10, Millipore, Billerica, MA, USA).

Solution properties

Collagen was dissolved in aqueous acetic acid. The concentration of collagen solution (5 mg mL⁻¹) and pectin (5 mg mL⁻¹) was used as a complex using acetic acid (50–80%). The reaction process is based on salt dependent mechanisms (NaCl). Solution conductivity is measured using a conductivity cell attached to an Orion benchtop multimeter (Thermo Fisher Scientific, Waltham, MA, USA).

The particle size variation of collagen–pectin microparticles are determined by dynamic light scattering (DLS) equipment (Delsa Nano C particle analyser, Beckman counter) with a He–Ne laser (632.8 nm, 35 mW) as light source. All the measurements are repeated three times. The particle size and the standard deviations obtained are used to fit the particle size distribution to a lognormal distribution. The zeta potential of the samples is determined by a Zeta Potential Analyzer from Delsa Nano C analyzer with Beckman counter. The zeta (ξ) potential is automatically calculated from electrophoretic mobility based on the Smoluchowski equation, v = (εE/η)ξ, where v is the measured electrophoretic velocity, η is the viscosity, ε is the electrical permittivity of the electrolytic solution and E is the electric field. When an electric field is applied to charged particles in the suspension, particles move towards an electrode opposite to their surface charge. Since the velocity is proportional to the amount of charge of the particles, zeta potential can be estimated by measuring the velocity of the particles.

Preparation of collagen–pectin microparticles

Collagen–pectin solutions (10 mg mL⁻¹) are supplied by a syringe pump for spraying from the grounded nozzle. Aluminum foil is placed perpendicular to the nozzle as a deposition target, to which a high negative voltage is applied. Solutions are dispersed into droplets by an electrical field generated between the nozzle and target. The solvent is evaporated before reaching the target, where the solid particles get collected. All preparations are carried out in an acrylic chamber at room temperature. The humidity is controlled to be lower than 20% RH by flowing nitrogen gas. Processing parameters are applied voltage, −25 kV; nozzle diameter, 0.65 mm; flow rate of the feed solution, 0.2 mL min⁻¹; distance between the nozzle and target H, 10 cm.¹⁷

Characterisation of collagen–pectin microparticles

FTIR analysis. Fourier-transform infrared (FT-IR) studies are carried out on collagen–pectin microparticles after the spraying process using an ATR-FTIR spectrophotometer (Jasco 6200, JASCO Corp, Tokyo, Japan). All spectra are recorded in absorption mode at 2 cm⁻¹ intervals.

Differential scanning calorimetry. Assessment of thermal stability of collagen–pectin microparticles is made by measuring the denaturation temperature (T_d). The change in T_d indicates alteration in collagen thermal stability. Thermal transitions of samples in collagen–pectin ratio group as a function of acetic acid concentration are measured using a TA Instruments DSC-Q2000 Differential Scanning Calorimeter (METTLER TOLEDO Company, Switzerland) over a temperature range of 25 to 150 °C at a heating rate of 5 °C min⁻¹. T_d is recorded at maximum peak height of the denaturation endotherm.¹⁸

X-ray diffraction analysis. X-ray diffraction (XRD) analysis is conducted using a D/Max-2550 PC X-ray diffractometer (Rigaku, Japan) with Cu K_α radiation for collagen–pectin complex prepared with neutrals salts (NaCl) as a function of acetic acid concentration (50–80%).^19,20

Scanning electron microscopy analysis. The morphologies of the collagen–pectin microparticles as electrosprayed powder are observed under a scanning electron microscope (SEM) (SU8000, Hitachi, Tokyo, Japan) at an accelerating voltage of 10 or 15 kV. Prior to scanning under the SEM, the samples are sputter coated for 30 s with platinum coater (E-1030 ion sputter, Hitachi, Tokyo, Japan). Particle size is determined through the analysis of SEM pictures using Mac View ver. 4.0 software (Mountech, Tokyo, Japan). Three hundred particles are selected randomly from the image to obtain the Heywood diameter. Volume-mean diameter is used for the analysis.²¹

AFM analysis. The size and surface topography of collagen–pectin microparticles is analyzed by Scanning probe microscope (JSPM 5200). Joel measurements is performed in air. For AFM analysis in air, the surface is air-dried in a dust-free enclosure for 24 h and is imaged in non-contact mode with a spring constant of about 20–100 N m⁻¹ and a resonance frequency of about 250–350 kHz.²¹

Antagonistic evaluation of collagen–pectin microparticles

Antimicrobial susceptibity test. The plate spreading method is used, which involves swabbing of cultures on to the surface of the solidified agar medium. Twenty four hours old cultures of different organisms are taken. A loopful of the culture is uniformly spread over the surface of a sterile Muller-Hilton agar with a sterile bent rod.²² collagen–pectin microparticles (100 μg mL⁻¹) prepared using different acetic acid concentrations such as 50–80% with the addition of NaCl (0.25%) using sterile peptone water. The prepared particles are used to fill hole bored by 5 mm cork borer in the inoculated agar. The plates are made in triplicate with one for the test organisms. All the plates are incubated at 37 °C for 24 h. The diameter of the zone of inhibition in the triplicate plates is measured by calculating the difference between core borer (5 mm) and the diameters of inhibition²³ and the mean representing their zone of inhibition.

Test for antibacterial activity of collagen–pectin microparticles

Turbidity measurement of bacterial growth is carried out using UV-visible spectrophotometer (Cary 100 Conc). The density of bacterial cells in liquid cultures is estimated by optical density (OD) measurements at 600 nm wavelength.

In vitro cytotoxicity analysis

Fibroblasts used in the experiments are isolated by the method described by Purna et al. (2001).^24,25 The cytotoxicity of collagenous matrices is evaluated by the MTT assay and cell morphology in contact with the tested sample. Fibroblasts at a concentration of 4 × 10⁴ cells per well are directly seeded into 12-well culture plates into which a collagen sample are placed and then cultured for 24 h. Cell morphology is observed by optical microscopy. The changes in the cell viability under conditions of co-culture with the tested samples are measured using the MTT assay. The well into which no tested matrix is placed is used as the control. At the end of culture, the yellow tetrazolium MTT solution is added and incubated for 3 h until a purple precipitate is visible.²⁶ The absorbance of each well is recorded at 600 nm.

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