Development and in vitro evaluation of oxytetracycline‐loaded PMMA nanoparticles for oral delivery against anaplasmosis

Abstract

Poly‐methyl methacrylate (PMMA) polymer with remarkable properties and merits are being preferred in various biomedical applications due to its biocompatibility, non‐toxicity and cost effectiveness. In this investigation, oxytetracycline‐loaded PMMA nanoparticles were prepared using nano‐precipitation method for the treatment of anaplasmosis. The prepared nanoparticles were characterised using dynamic light scattering (DLS), atomic force microscopy (AFM), differential scanning calorimetry (DSC) and Fourier transform infrared (FTIR) spectroscopy. The mean average diameter of the nanoparticles ranged between 190–240 nm and zeta potential was found to be −19 mV. The drug loading capacity and entrapment efficiency of nanoparticles was found varied between 33.7–62.2% and 40.5–60.0%. The in vitro drug release profile exhibited a biphasic phenomenon indicating controlled drug release. The uptake of coumarin‐6(C‐6)‐loaded PMMA nanoparticles in Plasmodium falciparum (Pf 3D7) culture model was studied. The preferential uptake of C‐6‐loaded nanoparticles by the Plasmodium infected erythrocytes in comparison with the uninfected erythrocytes was observed under fluorescence microscopy. These findings suggest that oxytetracycline‐loaded PMMA nanoparticles were found to be an effective oral delivery vehicle and an alternative pharmaceutical formulation in anaplasmosis treatment, too.

1 Introduction

Anaplasmosis, an infectious bacterial disease caused by Anaplasma marginale, poses serious health problems in cattle around the world [1, 2]. The infectious organism invades and destroys red blood cells, causing anaemia, weakness, and sometimes death [3]. Anaplasmosis is spread from infected to susceptible cattle by ticks (Ixodes scapularis) and biting insects [4, 5], which spread the disease by transmitting infected blood to susceptible animals. Infected cattles exhibit high body temperature (103–104°F) with increased lachrymation and salivation. Death often occurs within 24–48 h of the onset of symptoms and thereby signalling the disease among cattles in the herd [6]. Abortion is common even after recovery from clinical disease in many cases [7]. A death loss of 20–30% is more common and mortality rate may range between 5% and 10% in newly infected herds [8]. Young cattles may be off feed and moderately anaemic, but usually recover quickly with the passage of time. Cattles <1½ or 2 years of age are normally asymptomatic [9].

The causative organism A. marginale is susceptible to tetracycline such as oxytetracycline or chlortetracycline antibiotics when given in higher concentration [10, 11]. Unfortunately, cattles showing clinical signs usually respond to intermittent dosages of oxytetracycline given by injection [12, 13].Continued drug resistance in infectious organism(s) has become a challenging task in prevention and control of the disease. The antibiotics are not able to eliminate the organism once it is inside the erythrocyte. While in the circulation, tetracycline will have to wait until the causative organisms show up in the blood plasma. By that time, there would be a strong possibility that the tetracycline half‐life would be terminated and excreted via glomerular filtration [14] by allowing the infectious agent to escape and infect the new red blood cells. Hence, early recognition and treatment is crucial [15]. Generally, when clinical disease is diagnosed, immediate treatment of affected animals with high dosages (10 mg oxytetracycline per pound body weight for 3–5 days) is normally recommended [16]. In many herds, the cattles are fed with oxytetracycline at the rate of 0.1–0.25 mg or chlortetracycline at 0.5 mg per pound of body weight daily for 30 days throughout the vector season [13]. Although vaccines are available for anaplasmosis, which are limited to prevent the development of clinical disease, but not the infection from a herd [17]. Vaccinated cattle will remain carriers of infectious agent, even though they do not develop the disease themselves [18, 19].

A targeted drug delivery in controlled manner using nanotechnological approach may provide maximum therapeutic activity. This process would enable drug bioavailability, reduce dosing frequency, and may resolve the major obstacles in the control of intracellular pathogens. The cost effective strategy would reduce the burden to targeted farmers and the side effects in diseased animals. This article highlights the preparation, characterisation, and in vitro evaluation of oxytetracycline‐loaded nanoparticles formulated for the oral delivery.

2 Experimental methods

2.1 Materials

Poly (methyl methacrylate) (PMMA), oxytetracycline (OTC), poly vinyl alcohol (PVA), potassium bromide, DAPI (4′,6‐diamidino‐2‐phenylindole dihydrochloride), and coumarin‐6(C‐6) were purchased from Sigma‐Aldrich, Bangalore, India. DMEM, RPMI‐1640 and AlbuMAX II are Gibco products which were purchased from Invitrogen Corporation. Chloroform, methanol, acetone (AR grade) and phosphate‐buffered saline (PBS) was purchased from HiMedia Laboratories Pvt Ltd (Mumbai). Water used for all experiments was triple distilled water or Milli‐Q water. All other chemicals were of highest purity available and used without further purification.

2.2 Preparation of OTC‐loaded PMMA nanoparticles

PMMA nanoparticles in the size range of 190–240 nm were produced by nano‐precipitation method [20]. In a typical procedure, 20 mg of the PMMA polymer and a weighed amount of OTC were dissolved in 10 mL of acetone. The organic phase was added drop wise into aqueous phase containing 0.01% PVA as stabiliser under sonication (Sonics VC‐130PB, Vibra‐Cell™ Ultrasonic Processors, Newton, USA). The organic solvent was evaporated using magnetic stirrer under room temperature for 12 h. The nanoparticles were separated from the aqueous medium by centrifugation at 16,000 rpm for 20 min followed by washing twice with distilled water. Drug‐free nanoparticles were also prepared without addition of drug.

2.3 Separation of unincorporated drug from nanoparticles

The nanoparticles were separated by cross‐flow filtration using a Labscale tangential flow filtration (TFF) system (Millipore, India) fitted with Pellicon XL50 ultrafiltration cassette having 50,000 Da molecular weight cut‐off Biomax (Polyethersulfone) membrane to remove the free drug and unbound stabiliser. Filtration performed at feed pressure 2.07 bar (30 psi) and retentive pressure 0.69 bar (10 psi) by adding volumes of water collected as filtrate fractions. The amount of eliminated free oxytetracycline in each fraction was determined by using UV spectrophotometer at 264 nm.

2.4 Freeze‐drying

The prepared nano‐formulations were lyophilised in a freeze‐drier (Labconco Corporation, USA) under high vacuum (0.05 mbar) for 48 h. The dried samples were stored at −20°C prior to analysis. The nanoparticle yield was calculated as

$$\begin{array}{rl}& \mathrm{Nanoparticles}\mathrm{recovery}(\text{\%})\\ & =\frac{\mathrm{Mass}\mathrm{of}\mathrm{PMMA}\mathrm{nanoparticles}\mathrm{recovered}}{\mathrm{Mass}\mathrm{of}\mathrm{polymer},\mathrm{drug}\mathrm{and}\mathrm{stabilizer}}\times 100\end{array}$$

2.5 Physiochemical characterisation

2.5.1 Surface topography of nanoparticles

Topography of nanoparticles was studied with the atomic force microscope (AFM) using Nanoman (Veeco Instruments Inc., Plainview, NY, USA) at VINL, JNCASR, Bangalore. The nanoparticles were diluted in deionised water to an appropriate concentration (1 mg%) and then ultrasonicated in bath‐sonicator (Branson, USA) for 15–30 min. Two drops of the suspension were placed on a freshly cleaved mica surface, allowed to dry at room temperature and the images captured from the changes in vertical position using non‐contact AFM mode and silicon tips with spring constant of 20–80 N/m. Nanoparticle's surfaces and topography observed for the samples with high resolution by controlling the contrast.

2.5.2 Particle size and zeta potential

The average particle size and zeta potential of the nanoparticles were measured using a Zetasizer Nano ZS (Malvern Instruments Ltd, UK). About 20 μL of freshly prepared nanoparticle suspension was redispersed in 980 μL PBS and placed in a clear disposable zeta cell (DTS‐1060C). The measurements were carried out using a light source 4 mW HeNe laser (633 nm) at a fixed angle 173°. The following parameters were used for the experiments: medium refractive index 1.330, medium viscosity 0.8872 cP, a dielectric constant of 78.54, refractive Index of PMMA 1.49, absorption value of PMMA 0.06 and temperature of 25°C. All measurements were carried out in triplicate directly after nanoparticles preparation, and the results expressed as mean size ±SD.

2.5.3 Oxytetracycline content in the nanoparticles

Freeze‐dried nanoparticles were added into acetone and methanol (1:2) and centrifuged at 10,000 rpm for 10 min in cold centrifuge (Sigma, USA). The supernatant was analysed for OTC by reverse‐phase HPLC system (Shimadzu Corporation, Kyoto, Japan).

The mobile phase was 20 mM monopotassium phosphate (KH₂ PO₄), methanol and orthophosphoric acid (70:30:0.2, v/v/v). The HPLC system consisted of a model LC‐10AT_VP pumps and SPD‐10AV_VP UV detector (Shimadzu Corporation, Kyoto, Japan) linked to an injection valve with 20 μL sample loop. A reverse‐phase Lichrosphere^® C18 column (250 × 4 mm, 5 μm) from Merck was used. The analysis performed at a flow rate of 1.5 mL/min with the UV detector at 264 nm. Data acquisition and processing accomplished with a personal computer using Class VP software.

OTC content and entrapment efficiency calculated as

$$\begin{array}{rl}& \mathrm{Oxytetracycline}\mathrm{content}\left(\text{\%}\frac{W}{W}\right)\\ & =\frac{\mathrm{Mass}\mathrm{of}\mathrm{OTC}\mathrm{in}\mathrm{PMMA}\mathrm{nanoparticles}}{\mathrm{Mass}\mathrm{of}\mathrm{PMMA}\mathrm{nanoparticles}\mathrm{recovered}}\times 100\end{array}$$

$$\begin{array}{rl}& \mathrm{Entrapment}\mathrm{efficiency}\left(\text{\%}\frac{W}{W}\right)\\ & =\frac{\mathrm{Mass}\mathrm{of}\mathrm{OTC}\mathrm{in}\mathrm{PMMA}\mathrm{nanoparticles}}{\mathrm{Starting}\mathrm{mass}\mathrm{of}\mathrm{OTC}}\times 100\end{array}$$

2.5.4 Fourier transformed infrared spectra

Fourier transform infrared (FTIR) spectra (Spectrum Perkin Elmer RX 1) of OTC alone, OTC‐loaded PMMA nanoparticles, OTC‐free PMMA (blank) nanoparticles and PMMA polymer were recorded in potassium bromide pellets, and the spectrum was recorded between 4000 and 500 cm⁻¹ using a high‐energy ceramic source.

2.5.5 Thermal analysis

The thermal behaviour of the nanoparticles was analysed using a PERKIN‐ELMER Diamond DSC instrument (USA). Approximately, 5 mg of nanoparticles was accurately weighed into a 40‐μL hermetic aluminium pan and sealed. The samples were heated from 30 to 300°C at a rate of 50°C/min and readings recorded on the first heating ramp. Indium pan used as the reference standard to calibrate the temperature and energy scale of the apparatus. All experiments were carried out in triplicate.

2.5.6 In vitro release profile

Four millilitres of the nanosuspensions were placed in the dialysis bag (M _w cut‐off 12,000, Spectrum Medical Industries, Inc., USA). Hermetically sealed bag was immersed in 100 mL 0.1 N hydrochloric acid medium (pH 1.2) under sink conditions. The entire system kept at 37°C with continuous magnetic stirring at 200 rpm. At predetermined time points, an aliquot of the sample (1 mL) was drawn and added with fresh medium (1 mL) to maintain the volume. The calibration curve developed for OTC using HPLC‐UV at 264 nm and the experiments were repeated thrice.

2.5.7 In vitro uptake study

A. marginale was cultured using IDE8 cell lines in antibiotic‐free DMEM culture medium as suggested by earlier investigators [10]. Unfortunately, the IDE8 cell lines were overgrown by other contaminants and forced us to use gentamycin to establish the good growth of contaminants‐free IDE8 cell lines. Even then, the IDE8 cell line failed to show the expected confluence required to meet the uptake studies. Then, we preferred to look for the alternative model for our experimentation and settled with the P. falciparum, a malaria causative as it shares the host organism, biologically, in many ways.

In vitro culture of P. falciparum: The strain of P. falciparum used in the study was 3D7 (chloroquine sensitive) that was obtained from Suman Dhar's laboratory at the Special Centre for Molecular Medicine (Jawaharlal Nehru University, New Delhi, India). Heparinised whole O⁺ blood was collected from the Rotary Blood Bank, New Delhi, India, and red blood cells separated under sterile conditions by centrifugation to remove serum and buffy coat; and erythrocytes were washed two to three times using RPMI 1640 without AlbuMAX II (incomplete media). The strain was maintained by serial passages in human erythrocytes cultured at 4–5% haematocrit in RPMI‐1640 medium supplemented with 0.5% AlbuMAX II and gentamycin sulphate (hereafter referred as complete RPMI 1640) and incubated at 37°C under the atmosphere of mixed gases (5% CO₂, 5% O₂, and 90% N₂) in a plastic chamber. The levels of parasitaemia routinely monitored on blood smear with 5% Giemsa‐azure type B stain in phosphate buffer (20 mM, pH 7.2).

Preparation of C‐6‐loaded PMMA nanoparticles: In a typical procedure, 20 mg of the PMMA polymer and 100 µL 1 mg/mL of C‐6 were dissolved in 10 mL of acetone as organic phase. The C‐6 containing organic phase added drop wise into aqueous phase containing 0.01% PVA as stabiliser under sonication (Vibra‐Cell™ Ultrasonic Processors, Newton, USA). The organic solvent was evaporated using magnetic stirrer under room temperature overnight. The nanoparticles separated from the aqueous medium by centrifugation at 16,000 rpm for 45 min and washed twice with distilled water.

In vitro uptake of C‐6‐loaded PMMA nanoparticles in erythrocytes: We evaluated the intraerythrocytic uptake study on P. falciparum (3D7) in asynchronous culture. P. falciparum infected erythrocytes (5% haematocrit and 2% parasitaemia) was incubated at 37°C in the presence of various concentrations of C‐6‐loaded nanoparticles. The experiment was carried out using C‐6‐loaded PMMA nanoparticles in uninfected and infected erythrocytes. The intraerythrocytic uptake of the nanoparticles was determined at different intervals (5, 15, 30, 60, 120 and 240 min) of incubation with uninfected and infected erythrocytes by adding C‐6‐loaded PMMA nanoparticles of varying concentrations to the culture in the 24‐well plates. The incubation further continued at 37°C. During which whether the infected cells and uninfected cells (cultured separately) will allow uptake of C‐6‐loaded PMMA nanoparticles were observed. The culture medium from individual well was removed by careful suction, washed with fresh incomplete media by centrifuging at 2500 rpm for 5 min to remove the unbound C‐6‐loaded PMMA nanoparticles.

• (a) Quantitative analysis of intraerythrocytic uptake: The washed erythrocytes (fresh and Infected) were lysed by using 2:1 ratio of chloroform and methanol. The lysed sample was centrifuged at 10,000 rpm for 20 min to pellet the debris and then fluorescence intensity of supernatant containing C‐6 was determined at 460 nm excitation wavelength and 500 nm emission wavelength using Varian Cary Eclipse Fluorescence spectrophotometer. The observed values were recorded and plotted (Fig. 6 b) by comparing with the standard curve of C‐6 (Fig. 6 a). The experiment was repeated thrice.

• (b) Qualitative analysis of intraerythrocytic uptake: Thin smears were made from the washed erythrocytes, fixed with methanol and air‐dried. The smeared slides were added with 100 µL DAPI (1 ng/mL) and incubated in dark for 20 min. Excess DAPI was removed by repeated washing, and later wiped‐off the excess stain and air‐dried. The slides were observed under the inverted fluorescence microscope (NIKON Eclipse Ti‐U, Japan) using 100× magnification (Fig. 7). The experiment was repeated thrice to record the events.

Fig. 6.

Quantitative analysis of intraerythrocytic uptake

(a) Standard curve for C‐6 in 2:1CHCl₃ :methanol, (b) Quantitative uptake of C‐6 loaded PMMA nanoparticles with respect to time

Fig. 7.

Qualitative analysis of intraerythrocytic uptake of C‐6‐loaded PMMA nanoparticles

2.6 Statistical analysis

Results shown as mean ± SEM was compared with the Student's t ‐test and differences are considered significant at a level of p  < 0.05.

3 Results and discussion

3.1 Morphological evaluations

Atomic force microscopy (AFM) has proved to be a valuable tool to understand the surface morphology of polymeric nanoparticles during preparation and processing. We used the AFM to visualise the surface morphology of the freeze‐dried OTC‐free PMMA (blank) and OTC‐loaded PMMA nanoparticles before the in vitro drug release study. Figs. 1 a and b are the surfaces of the OTC‐loaded PMMA nanoparticles in 2D and 3D, respectively. It was evident from AFM images that PVA formed a matrix into which the nanoparticles were interspersed due to coat formation that surrounded the individual particles. It is observed that the nanoparticles appear to be spherical in shape and smooth surfaced (shown in Fig. 1). Surfaces of all PMMA nanoparticles were free from erosion confirming the greater satiability of PMMA nanoparticles enabling them to exhibit greater mobility, involve in water uptake and subsequent hydrolysis or degradation. This observation is in agreement with previous reports by other researchers [21, 22].

Fig. 1.

Atomic force micrograph of OTC‐loaded PMMA nanoparticles

(a) 2D view, (b) 3D view

3.2 Particle size and zeta potential

Nanoparticles were characterised for their mean particle diameter and size distribution. The average diameters, zeta potentials, pH values of OTC‐free nanoparticles and OTC‐loaded nanoparticles have been listed in Table 1. The average diameters of nanoparticles from different preparation ranged from 196 to 234 nm (Fig. 2). The polydispersity indices obtained from the measurements were around 0.15 or lower indicating narrow deviations in sizes [22]. As the in vivo fate of nanoparticles is primarily a function of their size and shape, which may also determine their toxicity.

Table 1.

Mean diameters (nm), zeta potentials (mV) and pH values of OTC‐free PMMA nanoparticles and OTC‐loaded PMMA nanoparticles

OTC‐free PMMA nanoparticles				OTC‐loaded PMMA nanoparticles
Diameters, nm	Polydispersity index	Zeta potential, mV	pH values	Diameters, nm	Polydispersity index	Zeta potential, mV	pH values
196.2 ± 2.31	0.136 ± 0.015	−5.23 ± 1.33	6.97	227.5 ± 7.56	0.140 ± 0.021	−19.45 ± 2.73	6.95

Fig. 2.

Size distributions and zeta potential measurement

(a) OTC‐free PMMA nanoparticles, (b) OTC‐loaded PMMA nanoparticles

The measurements from AFM revealed the particle size and shape of the nanoparticles (Fig. 1). The average sizes were similar to those reported by earlier studies involving nano‐precipitation method [23]. The zeta potential of the nanoparticles was negative. It is reported that high‐potential values would ensure a high‐energy barrier [24] and provide a good stability due to repulsive forces that prevent aggregation upon ageing [25]. Muller considered that a zeta potential of about −25 mV allows an ideal stabilisation of nanoparticles. Accordingly, −19 mV, which is the value obtained with OTC‐loaded PMMA nanoparticles favoured the best stability among the designed nanoparticles. The higher zeta potential value indicative of more the negative charge on the surface. This property would provide more stable particles in solution and less likely to aggregate and can be well suspended in water based solution suggesting an important application for in vitro and in vivo studies.

The cross‐flow filtration method adopted using a lab scale TFF system, which allowed the separation of both drug‐free and drug‐loaded nanoparticles from each other [26, 27]. The entrapment efficiency of OTC was higher in all the nano‐formulations. The per cent of theoretical encapsulation was 60.2%. Freeze drying, also known as lyophilisation, a non‐destructive method preferred to remove moisture achieved good yield and reduced manufacturing costs which are important for quality control and bio‐distribution studies in vivo. The conservation of a nanoparticle diameter size after freeze‐drying considered as a good indication [28]. Generally, contaminant water would adversely affect the physical and chemical stability, and to reach a shelf life of several years [29, 30, 31]. Freeze drying of OTC‐loaded PMMA nanoparticles and OTC‐free PMMA nanoparticles were recovered (∼80%) in dried form. During this process, we were able to notice enhanced hydrophobicity and dispersion of the freeze‐dried nanoparticles, which may be due to the presence of PVA as surfactant. The measurement of zeta potential is a promising method to evaluate the state of nanoparticles surface and to detect any eventual modification after freeze‐drying. For this reason, we used 5% sucrose as cryoprotectant and additional PVA to nanoparticles suspension before freeze‐drying resulted in decreased negative surface charge from −5.23 ± 1.33 mV in OTC‐free PMMA nanoparticles and −19.45 ± 2.73 mV in OTC‐loaded PMMA nanoparticles. This is because nanoparticles surface smeared due to hydrogen bonding between OH groups of the PVA and the surface of the nanoparticles.

3.3 Characterisation of the nanoparticles

3.3.1 Fourier transformed infrared spectra

Characterisation by FTIR spectroscopy especially carried out to determine the adsorption of the drug in the given nanoparticles. The advantage of FTIR over X‐ray crystallographic techniques is its unique capability to provide information on the structural details of a candidate molecule in solution with greater spatial and temporal resolution [32]. The FTIR has a specific basic principle, which governs the bonds and groups of bonds vibrate at characteristic frequencies. During analysis, the sample reflectance and transmittance of the infrared rays at different frequencies translated into an IR absorption plot and then analysed to match with known signatures of identified materials in the FTIR library. The FTIR spectrum of the OTC alone, PMMA alone, OTC‐free PMMA nanoparticles and OTC‐loaded PMMA nanoparticles clearly indicated the carbonyl group at 3020 cm⁻¹ (Fig. 3). There is little ease in these spectra except the peaks for the various C–H and C–O stretches and deformations (2952, 1440, 1215 and 1728 cm⁻¹). In addition to the peaks seen in the OTC‐free PMMA nanoparticles, a new peak with the OTC‐loaded PMMA nanoparticles observed at 1528 cm⁻¹ that corresponded to the NH₂ group of the drug and the peak at 1041 cm⁻¹ matching to the ketonic group of the drug, thus confirming the presence of OTC (Table 2). The peaks for the CH₃, CH₂ and O=C–O–C=O stretching appeared at a similar position as in the PMMA alone but with lower intensities. This could probably indicate the effect of polymer drug interactions leading to change in vibration energies of free groups. In addition, the decrease in intensity of the carbonyl group peak could also indicate its deviation from the free state.

Fig. 3.

FTIR analysis

(a) OTC alone, (b) PMMA polymer, (c) OTC and PMMA polymer admixed sample, (d) OTC‐loaded PMMA nanoparticles

Table 2.

FTIR peaks for OTC alone, PMMA polymer, OTC and PMMA polymer admixed sample, OTC‐free PMMA nanoparticle and OTC‐loaded PMMA nanoparticles

Wave numbers	OTC alone	PMMA polymer	OTC and PMMA admixed sample	OTC‐loaded PMMA nanoparticles	OTC‐free PMMA nanoparticles
3020	yes	yes	yes	yes	yes
2952	no	yes	yes	no	no
2401	yes	yes	yes	yes	no
2362	no	yes(week)	yes(week)	yes (moderate sharp)	yes (moderate sharp)
1728	no	yes	yes	yes	yes
1528	yes	no	yes	yes	no
1440	yes	yes	yes	yes	yes
1215	yes	yes	yes	yes	yes
1154	no	yes (intense)	yes (intense)	yes (week)	yes (week)
1041	yes	no	yes	yes	no
988	no	yes	yes	no	no
754	yes	yes	yes	yes	yes
670	yes	yes	yes	yes	yes

3.3.2 Differential scanning calorimetry

Differential scanning calorimetry (DSC) analysis is based on thermos‐gravimetric (Tg) data. From Fig. 4, there was no peak observed at the temperature of 50 and 550°C for the samples. The DSC study did not detect any crystalline drug material in the nanoparticles, suggesting that the OTC formulated in the samples was in amorphous or disordered‐crystalline phase of a molecular dispersion or a solid solution state in the polymer matrix after fabrication. The melting peak of OTC was absent in OTC‐free PMMA nanoparticles but present in OTC‐loaded PMMA nanoparticles. In Fig. 4, the glass‐transition temperature (Tg) of the OTC‐free PMMA nanoparticles was around 340°C. Tg of the OTC alone showed an exothermic peak at 210°C. However, this peak did not appear in the curves for the OTC‐loaded PMMA nanoparticles. The melting point of OTC in OTC‐loaded PMMA nanoparticles shown to be 210°C indicating that there was OTC in the sample. However, in an admixed sample containing PMMA polymer and free OTC the Tg of the polymer was diminished due to the presence of dissolved OTC in the formulation acting as a plasticiser [33]. This would suggest that a very small loading of OTC on to the PMMA matrix could significantly diminish the Tg of the PMMA polymer.

Fig. 4.

Differential scanning calorimetry analysis

(a) OTC alone, (b) OTC and PMMA polymer admixed sample, (c) OTC‐free PMMA nanoparticles, (d) OTC‐loaded PMMA nanoparticles

3.3.3 In vitro release profile

Initial burst release was significant and attributed to the immediate dissolution and release of OTC adhered on the surface and located near the surface of the nanoparticles. The initial rapid removal of the drug from nanoparticles possibly related to loss of loosely held drug on the surface of the nanoparticles. The release profile was optimised because the surfactant PVA employed plays a significant role in controlling the release profile [34, 35]. OTC‐loaded PMMA nanoparticles released around 50% of the drug within 4 h, which correlates with the onset of action. Later, the nanoparticles have shown significant sustained release effect by prolonging the release at 90.02 ± 0.01% till 25 h. The use of polymer PMMA has a significant effect on the drug‐sustained release over a prolonged time. This suggests in vitro drug release exhibited biphasic pattern by initial burst release followed by sustained release. The OTC‐alone was used to compare the effectiveness of the OTC‐loaded PMMA nanoparticles. The in vitro release profile of OTC from PMMA nanoparticles in 0.1 N HCl (pH 1.3) and 10% polyethylene glycol 400 (pH 6.9) solution is presented in Fig. 5. The PMMA polymers retain their hydrophobic properties on the surface of the nanoparticles, which get distorted in the acidic pH due to the protonation of carboxyl groups. Fig. 5 shows a very rapid diffusion of OTC from PMMA‐PEG diblock co‐polymers caused by swelling. As the pH shifts from acidic side to alkaline side, favouring carboxyl ionisation and hydrogen bond breakage. Surprisingly, 50% of OTC is release after 1.5 h and 100% after 8 h in comparison to the release in 0.1 N HCl exhibiting 50% of OTC is released in 1.5 h and 92% release after 12 h were observed. Drug release from nanoparticles is usually a biphasic phenomenon, although on some occasions a triphasic profile is also seen. The effect of acidic and near neutral pH conditions on OTC release seems to be marginal.

Fig. 5.

In vitro release profile of OTC‐loaded PMMA nanoparticles in 0.1 N HCl and 10% PEG 400

Gradual release of OTC was observed with increase in the time, which elevates the importance of sustained release property of the polymeric nanoparticles suggesting correlation with the increased solubility. The lower particle size of the nanoparticles had caused an increase in the effective surface area, which in turn increases the solubility [36]. This supports our rationale of increasing solubility, which may improve the uptake of candidate drug OTC by the infected erythrocytes either in vitro or in vivo.

3.3.4 In vitro uptake study

Biologically, the causative organism anaplasmosis shares the host organism of P. falciparum, a malaria causative in many ways. Several researchers have used C‐6 as an effective tool to visualise the uptake studies [37, 38, 39].

1. Quantitative analysis of intraerythrocytic uptake: The standard calibration curve was constructed by using different concentrations of C‐6 in 2:1 ratio of chloroform and methanol at 460 nm excitation wavelength and 500 nm emission wavelength using Varian Cary Eclipse Fluorescence spectrophotometer. This calibration curve allowed to accurately quantify the amount of C‐6 inside the erythrocytes after uptake. The intraerythrocytic uptake of the C‐6‐loaded nanoparticles was determined with both uninfected and infected erythrocytes, for uptake specificity. The purified C‐6 loaded nanoparticles were further analysed by comparing with standard curve of C‐6 (Fig. 6 a). The values obtained in this experiment revealed the preferential uptake of nanoparticles in infected RBC which may be due to the loss of integrity of cell membrane due to infection.

2. Qualitative analysis of intraerythrocytic uptake: It is a known fact that nanoparticles can gain entry into living cells. Cells are known to allow the nanoparticles to pass through them either endocytic mechanisms or passive entry via lipid bilayer. By using fluorescence microscopy, we attempted to observe the C‐6‐loaded PMMA nanoparticles gaining access through the cell membranes so that they are internalised by the erythrocytes with respect to time. Uninfected and Pf 3D7 infected erythrocytes were used to evaluate the efficiency of PMMA nanoparticles cellular uptake. Nucleus staining was performed using DAPI and observed under fluorescence microscope. PMMA nanoparticles cellular uptake progress is shown in Fig. 7 at different time intervals. It is evident based on the fluorescence (shown in Fig. 7), a significant fraction of the administered nanoparticles could be taken up by the infected erythrocytes. This suggests the morphological change of highly perforated membrane caused due to infection and such changes cannot be expected in uninfected erythrocytes. Therefore, C‐6‐loaded PMMA nanoparticles fabricated in this study could gain access through infected erythrocytes cell membranes and be internalised by non‐specific endocytic mechanisms or passive entry. The quantitative analysis indicated the moderate signals of C‐6 even in the uninfected erythrocytes. The fluorescence microscopic analysis of the slides revealed that there is visible uptake of C‐6 loaded PMMA nanoparticles in infected erythrocytes and no slides showed uptake in uninfected erythrocytes. The moderate fluorescence intensity observed in the qualitative study might be due to adherence of C‐6‐loaded PMMA nanoparticles on erythrocytes themselves, which warrants through study. However, the current investigation demonstrates that oxytetracycline‐loaded PMMA nanoparticles appear to be an effective oral delivery vehicle in anaplasmosis treatment, too.

4 Conclusion

The PMMA nanoparticles loaded with OTC, a sparingly water‐soluble anti‐anaplasmosis agent successfully prepared using PVA as emulsifier by nano‐precipitation method and characterised. In vitro release kinetics study shows that OTC‐loaded PMMA nanoparticles exhibited biphasic phenomenon indicating a slow and sustained release of cargo (almost 92% release at the end of 12 h in acidic pH). The UV peaks for OTC remained unchanged indicating retention of therapeutic activity in the formulation. Evaluation of OTC‐loaded PMMA nanoparticles formulations were found to be a potential and cost‐effective for sustained oral delivery system in terms of particle size distribution and optimum drug loading capacity characteristics. The C‐6‐loaded PMMA nanoparticles were just used as proof of cellular uptake of drug‐loaded PMMA nanoparticles through Pf 3D7 infected erythrocytes cell membrane. The results of C‐6‐loaded PMMA nanoparticle's intraerythrocytic uptake in infected erythrocytes is higher than that of uninfected erythrocytes. These results could be useful for predicting possible dose–response of OTC‐loaded PMMA nanoparticles in in vivo. Based on these studies, the formulations fabricated in this work could be promising formulations as oral delivery agent for treatment against anaplasmosis.