Subcutaneously Administered Ultrafine PLGA Nanoparticles

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Subcutaneously administered ultrafine PLGA nanoparticles containing doxycycline hydrochloride target lymphatic filarial parasites Yuvraj Singh, Adepu Srinivas, Mamta Gangwar, Jaya Gopal Meher, Shailja Misra-Bhattacharya, and Manish K Chourasia Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00206 • Publication Date (Web): 04 May 2016 Downloaded from http://pubs.acs.org on May 7, 2016

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Molecular Pharmaceutics

Subcutaneously administered ultrafine PLGA nanoparticles containing doxycycline hydrochloride target lymphatic filarial parasites Yuvraj Singh 1a, Adepu Srinivas 1a, Mamta Gangwar 1b, Jaya Gopal Meher a, Shailja MisraBhattacharyab, Manish K. Chourasiaa* a

Pharmaceutics Division, CSIR-Central Drug Research Institute, Lucknow, India, 226031 Parasitology Division, CSIR-Central Drug Research Institute, Lucknow, India, 226031

b

* Corresponding Author Manish K. Chourasia Sr. Scientist Pharmaceutics Division, CSIR-Central Drug Research Institute, Lucknow, India, 226031 Ph. No: +91 522-2772450, 2772550. Fax: +91 522 2623405 Email: [email protected] CSIR CDRI Communication XXX-2016-MKC 1 Authors contributed equally

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Abstract

Systemic chemotherapeutic targeting of filarial parasites is unfocussed due to their deep seated location in lymphatic vessels. This warrants prolonged dosing regimen in high doses for an anthelminthic like Doxycycline hydrochloride (DOX). In order to provide an alternative, we have constructed ultrafine PLGA nanoparticles of DOX (DPNPs), so as to exploit the peculiarity of lymphatic vasculature underneath sub cutaneous layer of skin, which preferentially allows entry of only 10-100nm sized particles. DPNPs were constructed using a novel solvent diffusion method aided by probe-sonication, which resulted in an average size 95.43±0.8 nm as per DLS, PDI 0.168±0.03, zeta potential -7.38±0.32, entrapment efficiency 75.58±1.94% and refrigerator stability of 7 days with respect to size in the optimized batch. TEM further substantiated the spherical shape of DPNPs along with their actual non hydrated size as being well below 100 nm. FTIR analysis of DOX, dummy nanoparticles and freeze dried DPNPs revealed that formulation step did not induce prominent changes in chemical nature of DOX. The drug release was significantly altered (p99%) was procured from Sigma Aldrich chemicals Pvt. Ltd (MO, USA). Triple distilled water used in all the experiments was prepared in a three-stage Millipore Milli-Q plus 185 purification system (Bedford, USA) and had a resistivity more than 18.2 mW/cm. A 0.22 µm cellulose membrane (Whatman International Ltd., Mailstone, England) was used for filtration of buffer. Parafilm (Parafilm “M” Laboratory Film, American Can Company, CT, and USA) was used for sealing tubes. All the solvents used were of HPLC grade. Drug free rat plasma was collected from healthy male Wistar rats provided by Laboratory Animal Services Division of CSIR-CDRI. Drug excipient compatibility studies via FT-IR spectroscopy The FTIR spectrums of pure DOX, DOX loaded PLGA nanoparticles (frieze dried DPNPs) and freeze dried dummy nanoparticles were obtained by using ATR FTIR-spectrometer (Agilent technologies). A total of 2 mg sample was placed under the probe and scanned at 4 mm/s at a resolution of 2 cm-1 over a wave number region of 400-4000 cm-1 using FTIR. The characteristics peaks of functional groups of blank PLGA nanoparticles, DOX in pure form as well as in formulation were determined, compared and possible interactions (if any) between drug and excipients were analyzed by obtained data. Preparation of DOX loaded PLGA nanoparticles (DPNPs)

PLGA nanoparticles were prepared by a new W/O/W double emulsion solvent diffusion technique for encapsulating hydrophilic molecules with minor changes11. Briefly, as per Table 1, a specified quantity of polymer (PLGA) and pluronic F 127 was dissolved in 3 mL ethyl acetate. To this organic solution, 1 mL triple distilled water containing known amount of DOX was added. The mixture was then sonicated over an ice bath using a probe sonicator (Sonics, USA) at 30 % amplitude for 2 min to form an W/O emulsion. The resulting primary emulsion was added to 9 mL triple distilled water and re-sonicated over an ice bath at 30% amplitude to form a W/O/W double emulsion. Ethyl acetate was eliminated by evaporation under reduced pressure, leading to hardening of PLGA around dissolved drug to form fully ripened nanoparticles (DPNPs). After determining entrapment efficiency (elaborated later) via a previously reported method12, free drug was removed via dialysis. Optimization studies were conducted to estimate the effect of formulation and processing variables including amount of polymer, surfactant concentration, sonication time, on size, zeta potential, entrapment efficiency and PDI of DPNPs. For comparative purposes, blank nanoparticles (PNPs), were also prepared similarly, with the exception of drug. The formulated nanoparticles were stored in cold refrigerated conditions until further use. Particle Size, Poly dispersity index (PDI) and zeta potential measurement

Average particle size, PDI and zeta potential of DPNPs was determined by Dynamic Light Scattering and Laser Doppler Anemometry (Zetasizer Nano-ZS, Malvern Instruments, UK). The nanoparticle formulations were diluted with triple distilled water for particle size and PDI analysis to obtain adequate correlation. Measurements were repeated for three equivalent



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batches. The stability of prepared DPNPs was evaluated by measuring particle size, zeta potential under different storage conditions like 4±2 °C for up to 4 months and at room temperature (25±2 °C) for 2 months. Table 1: Input parameters followed whilst developing DPNPs along with physicochemical characteristics of various formulations obtained during optimization. Data is represented as mean ± s.d. where number of sample equals 3.

Mean Size (nm)

PDI

Zeta Potential (mV)

5

68±3.64

0.164±0.03

-6.13 ±2.14

5.1±1.3

5

92.78±2.45

0.245±0.1

-7.79±1.98

14.2±1.5

Batch

Polymer (mg)

Drug Polymer Ratio

Surfactant (%)

Sonication (min)

S1

15

~1:1

3

S2

30

2:3

3

S3

60

1:3

3

5

100±4.03

0.203±0.04

-3.87±0.93

32.45±2.75

S4

90

1:4.5

3

5

108±3.58

0.257±0.08

-3.73±1.26

73.36±1.64

S5

120

1:6

3

5

S6

30

2:3

1

5

107.1±4.56

0.327±0.11

-2.84±1.2

15.43±2.43

S7

30

2:3

2

5

97.42±3.54

0.234±0.12

-3.30±1.6

16.28±1.76

S8

30

2:3

3

5

92.78±2.56

0.245±0.09

-7.79±0.9

17.83±1.1

%Entrapment Efficiency

Not Formed

S9

30

2:3

4

5

80.34±1.05

0.224±0.05

-4.1±0.36

15.14±2.30

S10

30

2:3

3

1.5

122.4±3.64

0.205±0.10

-6.58±1.8

13.24±4.29

S11

30

2:3

3

5

92.78±3.24

0.245±0.12

-7.79±1.1

16.85±2

S12

30

2:3

3

3

99.85±2.89

0.135±0.07

-1.52±1.3

17.18±2.3

S13

30

2:3

3

7.5

84.98±2.81

0.126±0.08

-2.27±0.2

15.76±1.9

S14

30

2:3

3

10

73.75±1.26

0.191±0.09

-1.57±0.2

19.32±3.2

S15

30

2:3

3

12.5

73.46±1.23

0.193±0.07

-3.84 ±1.5

11.76±3.9

10

95.43±0.8

0.168±0.03

-7.38±0.32

75.58±1.94

S16 90 1:4.5 3 PDI: Poly-dispersity index, s.d. values for n=3.

Analytical method

HPLC system was equipped with single LC 20 AT gradient pump (Shimadzu), a Rheodyne (Cotati, CA, USA) model 7125 injector with a 20 µL loop and SPD-20A AVP UV detector (Shimadzu). HPLC separation was achieved on a Purospher C18 column (250 mm, 4.6 mm, and 5 µm) (Merck). The acquired data was processed using Class VP software. The mobile phase consisted of acetonitrile: 0.1 % trifluoro acetic acid (TFA) (30:70) v/v flowing at a rate of 1.0 mL/min. Column effluents were monitored at 350 nm13. The mobile phase was degassed in a bath sonicator before use. Plasma samples from in-vivo pharmacokinetic experiment were extracted for DOX content by the following protocol. Accurately measured 250 µL of plasma samples were taken in a centrifuge tube and 1 mL methanol was added to them. The mixtures were vortexed vigorously for 2 min and subjected to centrifugation at 10000 rpm for 10 min. The clear supernatant was collected in glass centrifuge tubes and dried. The dried samples were reconstituted with methanol and injected in HPLC system for analysis. Determination of total drug content and entrapment efficiency Specified volume of formulation was dissolved in 1:1 mixture of ethyl acetate and methanol, and vortexed for 5 min. The mixture was thereafter diluted with methanol and analyzed chromatographically. Entrapment efficiency was determined by using Nanosep® centrifugal device which


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possesses two compartments divided by a 30 KDa molecular weight cut off ultrafilter (Pall Life Sciences, India). Accurately measured, 0.5 mL formulation was taken in upper compartment of Nanosep® and centrifuged at 12000 rpm for 30 min. Afterwards the filtrate was collected from the lower compartment and analyzed chromatographically as before. %Entrapement efficiency =

Total drug content − Free Drug × 100 TDC

Morphology and particle size measurement by transmission electron microscopy

The morphology and particle size of DPNPs were characterized by transmission electron microscopy. A drop of optimized nanoparticle suspension (S 16) was placed on a carbon film coated on a copper grid and flash frozen by purging in liquid nitrogen. The dried DPNPs were observed at 80 kV by using TECNAI G2 20 S- TWIN (FEI Netherlands) instrument and photomicrographs of different fields at several appropriate magnifications was taken 14. In-vitro drug release studies In-vitro drug release study for optimized DPNPs (S16) was conducted in triplicate by equilibrium dialysis membrane method. The amount of drug released from formulation was measured chromatographically as described before. The experiment was conducted by taking specified volume of formulation in a hermetically sealed dialysis membrane. The dialysis bag was then suspended in pH 7.4 phosphate buffer saline (PBS) at 37 °C being stirred at 100 rpm. Samples were collected at predetermined time points, with each sample withdrawal followed by an immediate media replenishment step to maintain sink conditions. The obtained dissolution curve was fitted into various kinetic release models such as zero-order, first-order, Korsmeyer–Peppas and Hixson–Crowell via linear regression method. This was done to excavate probable mechanism of drug release 15. In-vitro anti filarial activity Isolation of adult female worms Brugia malayi (B. malayi) was carried out by peritoneal washing of 120-180 days infected Meriones unguiculatus (jird) which were previously inoculated intraperitoneally by 200-250 infective larvae (L3). Concentration dependent invitro screening of DOX solution and DPNPs against adult female B. malayi was carried out in accordance with reported methods16. Briefly, actively motile female worms of B. malayi were placed in 48 well culture plates in duplicate containing 1 mL media (one female worm/mL/well). RPMI 1640 medium, fortified with 10% fetal bovine serum was used as a medium for parasites. Different concentrations (5, 10, 20, and 40 µg/mL) of drug and DPNPs were added to the culture plate and incubated in CO2 incubator at 37°C. The worms were examined under a stereo zoom microscope for up to 10 days and reduction in motility of parasite scored as 4+ (0%), 3+ (1-49%), 2+ (50-74 %), 1+ (75-99%), and D (100% reduction- death). For comparative purposes blank nanoparticles PNPs and PBS without drug was used as control. The adult worms were then processed for viability assay using 3-(4, 5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) dye as described earlier17 and absorbance was measured at 570 nm using a plate reader (Tecan, Untersbergstrasse, Grodig,



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Austria). Per cent inhibition in MTT reduction was calculated by comparing relative absorbance produced by worms housed in treated and untreated wells. From the obtained results 50% of inhibitory concentration (IC50) value of DOX and DPNPs was calculated. In-vivo pharmacokinetic studies In-vivo pharmacokinetic studies of DOX solution and drug loaded nanoparticles was conducted in Wistar rats weighing between 150-200 g. The animal experimentations were carried out following approved guidelines of Institutional Animal Ethics Committee. The animals were kept at 23–24 °C and 50–60 % RH with a normal 12 h light/dark cycle starting one week before the experiment. DPNPs and DOX solution were administered subcutaneously at a dose of 5mg/kg, by slightly drawing the ventral skin of rats in vicinity of their hind limbs, so as to avoid intramuscular entry. The rats were divided in to 2 groups each containing 6 animals, with the first group administered DOX solution (formed by dispersing DOX in PBS) and the second comparator group receiving equivalent dose of DPNPs. Serial blood samples (0.25 - 0.3 mL) were collected from retro orbital plexus of rats at predefined time points (0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, 36, and 48 hrs). Plasma was separated from collected blood by centrifugation at 3000 × g for 10 min and stored at -80 °C prior to analysis by HPLC. For lymphatic profiling two additional groups of rats containing 15 rats each were administered DOX solution and DPNPs subcutaneously as before. At each sampling point (2, 4, 8, 24 and 48 hrs), three rats from either group were euthanized and skinned. Thereafter, two superficial lymph nodes, namely subiliac lymph node lying in the fold of thigh musculature adjacent to the ileac artery and popliteal lymph node lying near biceps femoris in the hind limb were located and harvested 18. The selection of lymph nodes was decided on the basis of their anatomical vicinity to the site of formulation administration. To improve lymph node identification, to be euthanized rats were administered peanut oil via oral gavage one hour prior to their sacrificial time point. Lymph nodes collected at each time point were pooled separately, weighed, dipped in PBS and homogenised. The lymph homogenate was then treated as plasma and extracted for DOX and analysed via HPLC for drug concentration. The pharmacokinetic data and the derived parameters were calculated using WinNonlin (6.1; Pharsight, Mountain View, CA). In-vivo effect on microfilaraemia in B. malayi infected Mastomys coucha Male Mastomys of same age (6–8 weeks) were used as experimental animal model. These animals were fed on rodent diet and water ad libitum and housed in hygienic and standard conditions of light (12 h light/12 h dark) and temperature (∼28°C). L3 were recovered from Aedes aegypti fed on infected donor Mastomys 9 ± 1 day earlier 19. Infection in Mastomys was given by inoculation of 100 L3 of subperiodic strain of human lymphatic filariid B. malayi in the back region subcutaneously. After a harbouring period of 5–7 months, animals displaying B. malayi infection and an overtly progressive microfilria count were used in this study. The doxycycline regimen adapted here has been borrowed from a previously conducted study 20. A dosing interval of 72 hours was adapted on experimental groups of five


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animals each with DOX and DPNPs administered at a dose of 10mg/kg on days 0 (day of treatment initiation), 3, 6, 9, 12. Equal number of infected animals, either untreated or PNP treated were kept as control and vehicle control. Microfilariae (mf) count in 10 µl blood drawn from tail of animals between 12.00 p.m. and 13.00 p.m. was assessed just before treatment initiation, on day 10 post initiation of treatment (p.i.t.) and thereafter every 10th day till 120 days p.i.t. The animals were subsequently euthanized. In-vivo effect on microfilaraemia was expressed as per cent reduction in mf count over pre-treatment level 21, 22 . Macrofilaricidal and female sterilization efficacy

Adult worms were recovered from major organs of animals 120 days p.i.t (testes, lungs, lymph nodes and heart). Harvested organs were teased and the parasites recovered were examined microscopically to ascertain sex, levels of calcification, motility, mortality, cell adherence along with their number. Macrofilaricidal efficacy of DPNPs, DOX solution, PNPs and untreated control group was recorded as per cent change in adult worm (including male, female and total) recovery. The surviving females were teased individually in a drop of PBS to appraise the intrauterine embryonic stages of parasite. Number of sterile female worms recovered from the treated animals was compared with that of control animals and percent sterilization of female worms was calculated21, 23. Real time monitoring of Wolbachia load post treatment

The housekeeping wBm-ftsZ gene of Wolbachia was amplified from the genomic DNA pool of adult B. malayi worms and the amplified product was successfully cloned in pTZ57R/T vector. The vector was double digested by enzymes for confirmation of cloning/insertion. Gene insertion was also confirmed by PCR using colonies having recombinant vector. The plasmid was isolated from these positive colonies. Copy number of plasmid was calculated using formula given below and a standard curve was plotted 24. ) ! "#$%&' ( ,$*&+#*&= (% /01& 2 *3/$04 + 0"/&'6)(33093 × 2

"#+*&604& )/6.022 × 10>? %

In the current manuscript qRT-PCR assay was used to quantify the copy number of wBm-ftsZ housekeeping gene isolated from the total RNA of female worms recovered from infected M. coucha after 120 days p.i.t using the standard curve. RNA was isolated by TRIzol reagent (Invitrogen) and first strand cDNA synthesis kit (Sigma-Aldrich,USA) was used for the synthesis of cDNA from RNA which was further used as a template for real time PCR quantification. Statistical evaluation

Statistical appraisal of stability batches and results of worm recovery assay was carried out by applying one way analysis of variance (ANOVA) followed by Dunnett's Multiple Comparison Test using readings of day 1 as control group. Results other than worm recovery



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in the in-vivo anti-filarial assay were compared using one way ANOVA followed by Newman Kewls Post Test. Other statistical comparisons entailed two way ANOVA for comparing dissolution profiles and pharmacokinetic data and unpaired t-tests for comparing IC50 values with p

Subcutaneously Administered Ultrafine PLGA Nanoparticles

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