part ii: poly (ethylene sebacate) nanoparticles of anti...
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Part II: Poly (ethylene sebacate)
Nanoparticles of Anti HIV-Anti tubercular drug
combinations
Poly (ethylene sebacate) Nanoparticles of Anti HIV-Anti tubercular drug combinations
106 Particulate Carriers as Drug Delivery Systems for Anti-Tubercular and Anti-Cancer Agents
6.1. INTRODUCTION
Development of AIDS from acquisition of HIV infection to disease progression
represents a tremendous social, economical and political challenge in the 21st Century
[Strawford and Hellerstein, 1998]. Further existence of tuberculosis in HIV patients
results in significantly higher mortality. Treatment failures in HIV-infected patients
have been associated with reduced drug concentrations due to malabsorption of anti-
mycobacterials. Also the M. avium–M. intracellulare (MAC) complex is the main
cause of complications in immunodepressed patients. Patients with severe
immunodeficiency benefit from co-administration of anti-HIV and anti-tubercular
drug.
Highly active antiretroviral therapy (HAART), a combination therapy of at least three
antiretroviral drugs was a major step forward in the treatment of AIDS and has led to
a significant reduction in the mortality [Frezzini et al., 2005; Holtgrave, 2005; Piliero,
2004]. Lopinavir (LOPI) a potent protease inhibitor is an integral drug in Highly
Active Anti-Retroviral Therapy (HAART) and rifampicin is a first line anti-tubercular
drug. Administration of Lopinavir alone has insufficient bioavailability due to
extensive first pass effect and limited intestinal uptake due to p-glycoprotein efflux
however, its blood levels are greatly increased by subtherapeutic doses of ritonavir, a
potent inhibitor of cytochrome P450 3A4. Ritonavir acts as a pharmacokinetic booster
for lopinavir but at the same time it increases the cost and exposure associated toxicity
in the patients.
The therapeutic strategy for the treatment of AIDS and tuberculosis has undergone a
paradigm shift in the past decade, wherein targeted delivery of anti-HIV and anti-
tubercular drugs is emerging as a new dimension. Nanoparticulate based drug delivery
system represents an attractive carrier for targeted drug delivery to viral and bacterial
reservoirs [Vyas et al., 2006; Chellat et al., 2005; Gunaseelan et al., 2010; Lanao et
al., 2007; Gupta and Jain, 2010; Neves et al., 2010; Wong et al., 2010].
Our group has recently reported a new biodegradable polymer, polyethylene sebacate
(PES) which offers some unique advantages including ease of synthesis, hydrophobic
nature, good hydrolytic stability and low cost. Enzymatic degradation studies with
lipase revealed PES as biodegradable and toxicity studies including genotoxicity and
mutagenicity have confirmed safety of PES for biomedical and pharmaceutical
applications [Malshe et al., 2006; More et al., 2009]. FDA approved polymer PLGA
and PLA were selected for comparative evaluation. Although rifampicin is an inducer
Poly (ethylene sebacate) Nanoparticles of Anti HIV-Anti tubercular drug combinations
107 Particulate Carriers as Drug Delivery Systems for Anti-Tubercular and Anti-Cancer Agents
of cytochrome P450 we hypothesized that nanoparticulate drug delivery systems
(NPDDS) would enable improved bioavailability of lopinavir by providing protection.
The objective of the study is to evaluate the role of the NPDDS of rifampicin-
lopinavir combinations on enhanced bioavailability of the drugs following oral
administration. Entrapping both drugs in the ratio 1:1 is an additional objective.
6.2. LOPINAVIR
Lopinavir is a peptidomimetic HIV protease inhibitor that is structurally similar to
ritonavir but is three- to tenfold more potent against HIV-1 in vitro. Lopinavir is
active against both HIV-1 and HIV-2.
6.2.1. Chemical structure:
Formula - C37H48N4O5 Molecular weight – 628.80 g/mol
6.2.2. Chemical name- (2S)-N-[(2S,4S,5S)-5-[2-(2,6-dimethylphenoxy) acetamido]-
4-hydroxy-1,6-diphenylhexan-2-yl]-3-methyl-2-(2- oxo-1,3- diazinan-1-l)butanamide
6.2.3. CAS number - 192725-17-0
6.2.4. Physicochemical properties
Lopinavir is a white to light tan powder. It is freely soluble in methanol and ethanol,
soluble in isopropanol and practically insoluble in water. Lopinavir has melting point
of 120-124°C.
6.2.5. Analysis
Several chromatographic methods have been reported for analysis of lopinavir as a
bulk drug, in formulations and from the biological fluids. Reported methods include
those based on HPLC separation with ultra-violet detection [Kuschak et al., 2001;
Choi et al., 2007; Weller et al., 2007; Hirano et al., 2010] and liquid
chromatography/tandem mass spectroscopy [Rezk et al., 2008].
6.2.6. Indications (Qazi et al., 2002)
Lopinavir is indicated in combination with other antiretroviral agents for the treatment
of HIV-infection.
Poly (ethylene sebacate) Nanoparticles of Anti HIV-Anti tubercular drug combinations
108 Particulate Carriers as Drug Delivery Systems for Anti-Tubercular and Anti-Cancer Agents
6.2.7. Mechanism of action (Qazi et al., 2002)
Lopinavir inhibits the HIV viral protease enzyme. This prevents cleavage of the gag-
pol polyprotein and, therefore, improper viral assembly results. This subsequently
results in non-infectious, immature viral particles.
6.2.8. Pharmacokinetic properties (Cvetkovic and Goa, 2003)
Lopinavir is only available as a coformulation with low doses of ritonavir. When
administered orally without ritonavir, lopinavir plasma concentrations were
exceedingly low mainly owing to first-pass metabolism. Lopinavir is absorbed rapidly
after oral administration. A moderate-to high-fat meal increases oral bioavailability by
up to 50%, and it is therefore recommended that the drug be taken with food.
Although the capsules contain lopinavir–ritonavir in a fixed 4:1 ratio, the observed
plasma concentration ratio for these two drugs following oral administration is nearly
20:1, reflecting the sensitivity of lopinavir to the inhibitory effect of ritonavir on
CYP3A4. Lopinavir undergoes extensive hepatic oxidative metabolism by CYP3A4.
Approximately 90% of total drug in plasma is the parent compound, and less than 3%
of a dose is eliminated unchanged in the urine. Both lopinavir and ritonavir are highly
bound to plasma proteins, mainly to α1-acid glycoprotein, and therefore have a low
fractional penetration into cerebrospinal fluid (CSF) and semen.
6.2.9. Adverse drug reaction (Cvetkovic and Goa, 2003)
The most common adverse events reported with the lopinavir–ritonavir coformulation
have been gastrointestinal, including loose stools, diarrhea, nausea, and vomiting. The
most common laboratory abnormalities include elevated total cholesterol and
triglycerides. Because the same adverse effects occur with ritonavir, it is unclear
whether the side effects are due to ritonavir, lopinavir, or both.
6.2.10. Contraindications (Cvetkovic and Goa, 2003)
Numerous dosing schedules exist for the treatment of DOX depending on disease,
response and concomitant therapy. Guidelines for dosing also include consideration of
white blood cell count. Dosage may be reduced and/or delayed in patients with bone
marrow depression due to cytotoxic/radiation therapy.
6.2.11. Marketed formulations
Several marketed dosage forms of lopinavir as fixed dose combination with ritonavir
are available in the US and Indian market including: Kaletra® capsules (Abbott),
Kaletra® oral solution (Abbott), Kaletra® tablets (Abbott), Emletra tablets (Emcure),
Poly (ethylene sebacate) Nanoparticles of Anti HIV-Anti tubercular drug combinations
109 Particulate Carriers as Drug Delivery Systems for Anti-Tubercular and Anti-Cancer Agents
Lopimune tablets (Cipla), Ritocom tablets (Hetero HC) and V-Letra tablets
(Ranbaxy).
6.3. ANALYTICAL METHOD DEVELOPMENT
The following methods for the simultaneous analysis of rifampicin and lopinavir were
developed:
Stability indicating HPLC method
HPLC method for the simultaneous analysis of rifampicin and
lopinavir in plasma and organ homogenate
6.3.1. Stability indicating HPLC method
Instrumentation:
The HPLC system used was JASCO LC900 Intelligent pump coupled with UV
detector (Jasco UV/VIS 1570/1575) and Rheodyne injector model (7725) fitted with
100μl sample loop. Data integration was done by Borwin chromatography software
version 1.21.
Chromatography
Chromatography was performed on a Waters Spherisorb® S5 ODS2 (250 × 4 mm i.d.,
5 μm particle size) column. The mobile phase comprised of tetrahydrofuran:methanol:
phosphate buffer pH 5.2 in the ratio 30:30:40 v/v was used. The mobile phase was
filtered through a nylon membrane (0.22 μm, Pall Gelman) and degassed by
sonication prior to use. Chromatography was performed at room temperature under
isocratic conditions at a flow rate of 1 mL/min. UV detection was done at a λmax of
212 nm.
Preparation of standard solutions
Rifampicin (10mg) and lopinavir (10mg) was accurately weighed and transferred to a
10 mL volumetric flask. The volume was made up to 10 mL with methanol to obtain
a stock solution (1000μg/mL). From the above solution, 0.05 and 0.1 ml was diluted
upto 10 ml with mobile phase to get concentration of 5 and 10μg/mL. Aliquots of
10μg/mL solution corresponding to 0.01, 0.05, 0.1, 0.5, and 1.0mL were diluted to
10mL with mobile phase to get concentration range from 10 to 1000ng/mL. Each
solution was injected in triplicate. Average of the peak areas was considered for
calculation purposes.
Poly (ethylene sebacate) Nanoparticles of Anti HIV-Anti tubercular drug combinations
110 Particulate Carriers as Drug Delivery Systems for Anti-Tubercular and Anti-Cancer Agents
Validation
a) Stability of analyte in solution:
The stability of rifampicin and lopinavir combination in mobile phase was assessed by
injecting the standard solution (10μg/ml) at interval of 0, 3 and 6 hrs post preparation
kept in amber colored volumetric flasks at room temperature. The chromatograms
were checked for presence of peaks corresponding to degraded product.
b) Linearity
Standard solutions (10, 50, 100, 500, 1000 ng/mL, 5 and 10μg/ml), each in three
replicates, were injected into the system. The method of linear regression was used for
data evaluation. Peak areas were plotted against theoretical concentrations of
standards. Linearity was expressed as a correlation coefficient.
c) Precision
System precision (repeatability) was determined by performing five consecutive
injections of the 10 μg/ml standard solution. Method precision was determined by
injecting three different samples (10 μg/ml) prepared individually.
Forced Degradation Studies
It was necessary to perform forced degradation studies to verify and prove the
stability-indicating feature of the proposed method. Intentional degradation was
attempted by heating the drug in the presence of base, acid and hydrogen peroxide
and exposing to sunlight.
i) Acid degradation
To 1mL of standard stock solution (100μg/mL) of rifampicin and lopinavir
combination, 1 mL of 0.1N HCl was added and the solution was placed in a boiling
water bath for 2h. The sample was allowed to cool to room temperature and
neutralized using 0.1N NaOH. The volume was adjusted to 10 ml with mobile phase,
and this solution was injected in the HPLC column.
ii) Base degradation
To 1mL of standard stock solution (100μg/mL) of rifampicin and lopinavir
combination, 1 mL of 0.1N NaOH was added and the solution was placed in a boiling
water bath for 2h. The sample was allowed to cool to room temperature and
neutralized using 0.1N HCl. The volume was adjusted to 10 ml with mobile phase,
and this solution was injected onto the HPLC column.
Poly (ethylene sebacate) Nanoparticles of Anti HIV-Anti tubercular drug combinations
111 Particulate Carriers as Drug Delivery Systems for Anti-Tubercular and Anti-Cancer Agents
iii) Oxidation
To 1mL of standard stock solution (100μg/mL) of rifampicin and lopinavir
combination, 1 mL of 3% H2O2 was added and the solution was placed in a boiling
water bath for 2h. The sample was allowed to cool to room temperature. The volume
was adjusted to 10 ml with mobile phase, and this solution was injected onto the
HPLC column.
iv) Photodegradation
Drug solution (10μg/mL) in mobile phase was exposed to sunlight for 2h and this
solution was injected onto the HPLC column.
The degraded samples were analyzed against an untreated control sample.
Results and Discussion
a) Stability of analyte in solution
Rifampicin and lopinavir was found to be stable in the mobile phase, when the
standard solution of strength 10μg/mL was analyzed at 0-6h post preparation. No
peaks corresponding to the degradation products were observed. A low RSD value
indicated that there was no significant change in the drug peak area (table 6.1).
Table 6.1: Stability of rifampicin and lopinavir in mobile phase
Rifampicin Area 1 Area 2 Average %RSD
0 hr 1373917 1363530 1368724 0.5366 3 hr 1367295 1378098 1372697 0.5564 6 hr 1358894 1368932 1363913 0.5205
AVG. 0.5378 Lopinavir Area 1 Area 2 Average %RSD
0 hr 2390291 2410153 2400222 0.5851 3 hr 2367945 2418125 2393035 1.4827 6 hr 2395331 2429923 2412627 1.0138
AVG. 1.0272
b) Linearity
Graph of the peak area vs concentration was plotted in order to check the linearity.
The developed method was found to be linear between concentration range of 0.01-
10μg/mL (figure 6.1 and 6.2). The regression coefficient was found to be 1 for both
RIF and LOPI.
Poly (ethylene sebacate) Nanoparticles of Anti HIV-Anti tubercular drug combinations
112 Particulate Carriers as Drug Delivery Systems for Anti-Tubercular and Anti-Cancer Agents
Standard Curve of Rifampicin
y = 136230x + 3546.4R2 = 1
0200000400000600000800000
1000000120000014000001600000
0 2 4 6 8 10 12Concentration mcg/ml
Are
a un
der
curv
e
Figure 6.1: Standard curve of rifampicin by HPLC
Standard Curve Lopinavir
y = 239530x - 3320R2 = 1
0
500000
1000000
1500000
2000000
2500000
3000000
0 2 4 6 8 10 12Concentration mcg/ml
Are
a un
der
curv
e
Figure 6.2: Standard curve of lopinavir by HPLC
c) Precision
Low RSD values of 0.35% for RIF and 0.72% for LOPI for system precision and for
method precision 0.72% for RIF and 0.50% for LOPI were obtained (table 6.2).
Table 6.2: Precision study of the assay
Sample No.
Area for Rifampicin Area for Lopinavir System
precision (10 µg/ml)
Method precision (10 µg/ml)
System precision (10 µg/ml)
Method precision (10 µg/ml)
1 1363530 1373917 2388912 2390291 2 1364972 1363530 2430674 2410153 3 1369832 1354160 2391234 2387991 4 1359454 - 2415397 - 5 1371298 - 2410164 -
% RSD 0.3522 0.7246 0.7243 0.5085
Forced Degradation Studies
The HPLC procedure was optimized with view to develop a stability indicating
method so as to resolve the degraded products from the drugs. Various mobile phase
Poly (ethylene sebacate) Nanoparticles of Anti HIV-Anti tubercular drug combinations
113 Particulate Carriers as Drug Delivery Systems for Anti-Tubercular and Anti-Cancer Agents
compositions were tried so as to obtain a sharp peak and also resolve the peaks of
degraded product from the peak of drug. The mobile phase consisting of
tetrahydrofuran:methanol: phosphate buffer pH 5.2 in the ratio 30:30:40 v/v resulted
in a retention time of 3.7 min for rifampicin and 9.5 min for LOPI. The
chromatograms of rifampicin and LOPI (undegraded) and rifampicin and LOPI
degraded in the presence of acid, base, hydrogen peroxide (H2O2) (oxidative
degradation) and light (photo degradation) are shown in figure 6.3 and indicated a
good separation of the undegraded drug from the degradation products. The peaks of
all the degraded products were resolved from the rifampicin and LOPI peak. The
chromatograms after forced degradation are shown in figure 6.3.
a) Standard rifampicin and LOPI
b) Acid degraded rifampicin and LOPI
c) Base degraded rifampicin and LOPI
Poly (ethylene sebacate) Nanoparticles of Anti HIV-Anti tubercular drug combinations
114 Particulate Carriers as Drug Delivery Systems for Anti-Tubercular and Anti-Cancer Agents
d) H2O2 degraded rifampicin and LOPI
e) Photo degraded rifampicin and LOPI
Figure 6.3: Forced degradation studies of RIF and LOPI
Conclusion: The RP-HPLC method developed for rifampicin and Lopinavir
combination was found to be precise, rapid, accurate, and stability indicating. Thus
the method could be used for determining the stability of rifampicin and lopinavir.
6.3.2. HPLC method for plasma and organ homogenate
Instrumentation
The HPLC system used was JASCO LC900 Intelligent pump coupled with UV
detector (Jasco UV/VIS 1570/1575) and Rheodyne injector model (7725) fitted with
100μl sample loop. Data integration was done by Borwin chromatography software
version 1.21.
Chromatography
Chromatography was performed on a Agilent ZORBEX SB-C18 (250 × 4 mm i.d.,
5μm particle size) column. The mobile phase comprised of Acetonitrile: Ammonium
formate buffer pH 3.8 in the ratio 48:52 v/v was used. The mobile phase was filtered
through a nylon membrane (0.22 μm, Pall Gelman) and degassed by sonication prior
to use. Chromatography was performed at room temperature under isocratic
conditions at a flow rate of 0.8 mL/min. UV detection was done at a λmax of 212 nm.
Poly (ethylene sebacate) Nanoparticles of Anti HIV-Anti tubercular drug combinations
115 Particulate Carriers as Drug Delivery Systems for Anti-Tubercular and Anti-Cancer Agents
Preparation of standard solutions
Rifampicin (10mg) and lopinavir (10mg) was accurately weighed and transferred to a
10 mL volumetric flask. The volume was made up to 10 mL with methanol to obtain
a stock solution (1000μg/mL). From the above solution, 0.1 ml was diluted upto 10 ml
with mobile phase to get concentration of 10μg/mL. Aliquots of 10μg/mL solution
corresponding to 0.1, 0.25, 0.5, 1.0 and 5.0mL were diluted to 10mL with mobile
phase to get concentration range from 100 to 5000ng/mL in the presence of plasma
and various organ homogenate (lung, liver, spleen and kidney). Each solution was
injected in triplicate. Average of the peak areas were considered for calculation
purposes.
Recovery/extraction from plasma and organ homogenate
The recovery of RIF and LOPI from plasma and various organ homogenate (lung,
liver, spleen and kidney) was determined at five different concentrations namely 100,
250, 500, 1000 and 5000 ng/mL (n=3). To determine drug extraction efficiency of
method from plasma and various organ homogenate (lung, liver, spleen and kidney),
RIF-LOPI solution (100µL) (1–50µg/mL) was spiked to drug-free plasma and various
organ homogenate (lung, liver, spleen and kidney) (400µL) and vortexed vigorously
for 2 min followed by addition of equal volume (500µL) of methanol. The resulting
mixture was vortexed vigorously for 2 min and centrifuged at 20,000 rpm for 20 min
at 25 ºC. The supernatant was injected into the HPLC system. Extraction efficiency of
RIF-LOPI from plasma and various organ homogenate (lung, liver, spleen and
kidney) was calculated by comparing the peak heights of standard RIF-LOPI solution.
Validation
The chromatographic method was validated for linearity, specificity, sensitivity,
precision, and accuracy.
i) Linearity: All validation runs were performed in triplicate to assess variation.
Calibration curves were constructed in presence of plasma and various organ
homogenate (lung, liver, spleen and kidney) over the concentration range 100–
5000ng/mL for RIF-LOPI combination.
ii) Sensitivity: Limit of quantification (LOQ) of standard drug and spiked plasma and
various organ homogenate (lung, liver, spleen and kidney) was determined at a signal
to noise ratio of 1:10.
iii) Precision: System precision (repeatability) was determined by performing five
consecutive injections of the 5μg/mL for RIF-LOPI combination. Method precision
Poly (ethylene sebacate) Nanoparticles of Anti HIV-Anti tubercular drug combinations
116 Particulate Carriers as Drug Delivery Systems for Anti-Tubercular and Anti-Cancer Agents
was determined by injecting three different samples of 5μg/mL for RIF-LOPI
combination prepared individually.
Results and Discussion
The developed method showed good resolution of the drug with retention time (RT)
of 5.8 mins (RIF) and 26.5 mins (LOPI) for plasma and various organ homogenate
(lung, liver, spleen and kidney) with no interference. The chromatograms indicate that
RIF and LOPI peaks are well separated from other peaks in plasma and various organ
homogenate (figure 6.4). The HPLC analytical method was found to be linear
between concentration range of 100-5000ng/mL in the presence of plasma and organ
homogenate with high correlation coefficient of (table 6.3 and 6.4).
Blank plasma RIF-LOPI extracted from Plasma
Blank lung homogenate RIF-LOPI extracted from lung homogenate
Blank spleen homogenate RIF-LOPI extracted from spleen homogenate
Blank kidney homogenate RIF-LOPI extracted from kidney homogenate
Blank liver homogenate RIF-LOPI extracted from liver homogenate
Figure 6.4: Chromatogram of RIF and LOPI from biological samples
Poly (ethylene sebacate) Nanoparticles of Anti HIV-Anti tubercular drug combinations
117 Particulate Carriers as Drug Delivery Systems for Anti-Tubercular and Anti-Cancer Agents
Extraction efficiency/recovery was greater than 85% (table 6.3 and 6.4) for both RIF
and LOPI and low RSD values (<5%) were obtained for system precision (table 6.5
and 6.6).
Table 6.3: Linearity and recovery data of RIF from plasma and organ homogenate
Medium Linearity range Slope Intercept r2
Extraction efficiency/ recovery
Standard RIF 100-5000 ng/ml 201.5 26139 0.9990 -
Plasma 100-5000 ng/ml 224.5 39973 0.9980 97.32±7.73
Lung Homogenate
100-5000 ng/ml 160.6 14022 0.9990 89.96±6.78
Spleen Homogenate
100-5000 ng/ml 174.2 22172 0.9990 91.23±4.81
Liver Homogenate
100-5000 ng/ml 183.7 25876 0.9994 90.26±4.15
Kidney Homogenate
100-5000 ng/ml 195.2 28954 0.9995 91.12±3.68
Table 6.4: Linearity and recovery data of LOPI from plasma and organ homogenate
Medium Linearity range Slope Intercept r2
Extraction efficiency/ recovery
Standard LOPI 100-5000 ng/ml 300.8 5297 1.0000 -
Plasma 100-5000 ng/ml 358.2 21226 0.9990 98.71±3.10
Lung Homogenate
100-5000 ng/ml 294.4 19748 0.9970 98.61±1.37
Spleen Homogenate
100-5000 ng/ml 322.5 4310 1.000 100.28±3.84
Liver Homogenate
100-5000 ng/ml 312.3 10823 0.9994 89.86±5.59
Kidney Homogenate
100-5000 ng/ml 302.5 14352 0.9995 92.52±6.83
Poly (ethylene sebacate) Nanoparticles of Anti HIV-Anti tubercular drug combinations
118 Particulate Carriers as Drug Delivery Systems for Anti-Tubercular and Anti-Cancer Agents
Table 6.5: System precision data of RIF from plasma and organ homogenate
Sample No. Area (5000 ng/mL)
Plasma Lung Spleen Kidney Liver 1 1055963 985394 932481 1006862 980579 2 1096205 952783 962086 1010868 1006885 3 1110935 990182 955479 1019832 1012139 4 1052762 1009823 910862 1010024 1009856 5 1058235 973422 920973 1010023 1016732
Average 1074820 982320.8 936376.2 1011522 1005238 RSD 2.49 2.14 2.34 0.48 1.41
Table 6.6: System precision data of LOPI from plasma and organ homogenate
Sample No. Area (5000 ng/mL)
Plasma Lung Spleen Kidney Liver 1 1749988 1629056 1637075 1609423 1600352 2 1775248 1593497 1614940 1597271 1609842 3 1801976 1528615 1577209 1560862 1610239 4 1775737 1583723 1609741 1600487 1619581 5 1779823 1612986 1608627 1509892 1611929
Average 1776554 1589575 1609518 1575587 1610389 RSD 1.03 2.41 1.33 2.61 0.42
HPLC method was validated for linearity, specificity, sensitivity & precision thus
establishing that the method can be efficiently used for RIF-LOPI analysis in plasma
and various organ homogenate (lung, liver, spleen and kidney).
Conclusion: The RP-HPLC method developed for RIF-LOPI was found to be
specific, precise, rapid and accurate. Thus the method could be efficiently used for
RIF-LOPI analysis in plasma and various organ homogenate (lung, liver, spleen and
kidney).
6.4. EXPERIMENTAL METHODS
6.4.1. Materials
Poly (ethylene sebacate) [PES] was synthesized in our laboratory (Mw= 11300),
rifampicin (RIF), lopinavir (LOPI), poly vinyl alcohol (PVA), and trehalose 100
(Hayashibara Co. Ltd., Japan) were kindly gifted by Maneesh Pharma (Mumbai,
India), Hetero Drugs Pvt. Ltd.(Hyderabad, India), Colorcon Asia Pvt ltd and Gangwal
Chemicals Pvt. Ltd. (Mumbai, India) respectively. PLGA 50:50 (PDLG 5010;
inherent viscosity midpoint of 1 dl/g) was purchased from PURASORB®. Lutrol-F-68
Poly (ethylene sebacate) Nanoparticles of Anti HIV-Anti tubercular drug combinations
119 Particulate Carriers as Drug Delivery Systems for Anti-Tubercular and Anti-Cancer Agents
(polyoxyethylene polyoxypropylene block co-polymer) was a gift sample from BASF.
Dichloromethane AR, methanol AR, dioctyl sodium sulphosuccinate AR (Aerosol
OT®, AOT), tetrahydrofuran AR (THF), ammonium format AR, disodium hydrogen
phosphate AR and sodium chloride AR were purchased from s. d. fine-chem limited
(Mumbai, India). Ethyl alcohol AR (99.9% pure) was purchased from Changshu
Yangyuan Chemical (China). Filtered (0.45 µ membrane filter) doubled distilled
water was used for preparation of nanoparticles. All other chemicals and solvents
were either spectroscopic or analytical grade.
6.4.2. Preparation and optimization of nanoparticles
RIF-LOPI loaded PES nanoparticles were prepared by emulsion solvent evaporation
method. Briefly, RIF (25-35mg), LOPI (15-25mg) and PES (100mg) were dissolved
in dichloromethane (5ml). The non solvent phases comprised an aqueous solution of
poly vinyl alcohol (75mg) in 20ml water. The organic phase was added to the non
solvent phase by probe sonication for 5minutes (10sec on/10sec off cycle) to form a
stable emulsion. The dispersion was kept under continuous stirring on a magnetic
stirrer at room temperature till complete evaporation of organic solvent (approx. 2-
3h). The nanoparticle suspension was centrifuged at 15000 rpm for 30 min and the
supernatant analyzed for drug to determine entrapment efficiency. Similarly by
replacing PES with PLGA and PLA, RIF-LOPI loaded nanoparticles of PLGA and
PLA were prepared. Parameters evaluated included concentration of polymer,
concentration of surfactants and rifampicin to lopinavir ratio, to optimize particle size
and entrapment of both drugs in 1:1 ratio.
a) Effect of PVA concentration
Table 6.7: Effect of PVA concentration on %EE and particle size
RLPES/1 RLPES/2 RLPES/3
Rifampicin (mg) 25 25 25
Lopinavir (mg 25 25 25
PES (mg) 100 100 100
Dichloromethane (ml) 5 5 5
Water (ml) 20 20 20
Poly vinyl alcohol (mg) 50 (0.25% w/v)
75 (0.375% w/v)
100 (0.5% w/v)
Poly (ethylene sebacate) Nanoparticles of Anti HIV-Anti tubercular drug combinations
120 Particulate Carriers as Drug Delivery Systems for Anti-Tubercular and Anti-Cancer Agents
The surfactant concentration was varied as 0.25, 0.375 and 0.5% in order to find the
ratio that gives optimum entrapment efficiency table 6.7.
b) Effect of rifampicin to lopinavir ratio
The rifampicin to lopinavir ratio was varied to find the ratio that gives optimum
entrapment efficiency of both drugs in 1:1 ratio (table 6.8).
Table 6.8: Effect of RIF:LOPI ratio on %EE and particle size
RLPES/4 (1:0)
RLPES/5 (7:3)
RLPES/6 (6:4)
RLPES/2 (1:1)
Rifampicin (mg) 50 35 30 25 Lopinavir (mg - 15 20 25 PES (mg) 100 100 100 100 Dichloromethane (ml) 5 5 5 5 Water (ml) 20 20 20 20 Poly vinyl alcohol (mg) 75 75 75 75
c) Effect of drug: polymer ratio
Effect of RIF-LOPI:PES ratio (drug:polymer) on entrapment efficiency and particle
size was evaluated. Ratio of RIF-LOPI:PES evaluated include 1:1, 1:1.5, 1:2 and 1:3
(table 6.9).
Table 6.9: Effect of drug:polymer ratio on %EE and particle size
RLPES/8 (1:1)
RLPES/9 (1:1.5)
RLPES/7 (1:2)
RLPES/10 (1:3)
Rifampicin (mg) 32.5 32.5 32.5 32.5 Lopinavir (mg 17.5 17.5 17.5 17.5 PES (mg) 50 75 100 150 Dichloromethane (ml) 5 5 5 5 Water (ml) 20 20 20 20 Poly vinyl alcohol (mg) 75 75 75 75
6.4.3. Freeze-drying of nanoparticles
Freeze-drying of various nanoparticle batches were carried out using trehalose (10:1
by weight of nanoparticles) and lutrol-F-68 (0.1:1 by weight of nanoparticles).
Poly (ethylene sebacate) Nanoparticles of Anti HIV-Anti tubercular drug combinations
121 Particulate Carriers as Drug Delivery Systems for Anti-Tubercular and Anti-Cancer Agents
Samples of 10 mL dispersion of nanoparticles were dispensed in 250mL freeze drying
glass vessels, frozen at -70 °C for 12 h and then subjected to freeze-drying using
Labconco freeze-drying system (FreeZone 4.5, USA). Sublimation lasted for 36-48 h
at a vacuum pressure of 10-50×10-3 bar, with the condenser surface temperature
maintained at less than -50 °C. Lyophilized samples were collected under anhydrous
conditions and stored in a dessicator until re-hydrated. Re-hydration of lyophilized
nanoparticles was carried with 0.2 µm filtered water by simple manual shaking.
Particle size and PI of the re-hydrated samples were determined by PCS to assess the
cryoprotection provided by the cryoprotectant.
6.4.3. Evaluation and physical characterization
a) Entrapment Efficiency
Nanoparticle dispersion was centrifuged at 15,000 rpm for 30 min at 20 ºC. The
resultant supernatant was analyses for free drug. The concentration of RIF and LOPI
in the supernatant was determined using stability indicating HPLC method reported in
section 6.3.1. Entrapment efficiency was calculated using equation 6.
%EE = (RIF/LOPIinitial – RIF/LOPIsupernatant) / RIF/LOPIinitial ×100 (6)
b) Particle Size
Particle size was determined by Photon Correlation Spectroscopy using N4 plus
submicron particle size analyzer (Beckman Coulter, USA). The analysis was
performed at a scattering angle of 90º at a temperature of 25 ºC. All the
nanoparticulate dispersions were sonicated using ultrasonic probe system (DP120,
Dakshin, Mumbai, India) for 5 min with 10 sec pulse at 200 voltages over an ice bath.
Dispersions were then appropriately diluted with filtered water (0.2 µm filter,
Millipore India Pvt. Ltd.) to obtain 5 ×104 to 1×106 counts per second. Each sample
was analyzed in triplicate and average particle size and polydispersity index (PI)
measured.
c) Drug loading
Measured quantity of freeze dried nanoparticles were dissolved in THF:water (1:1) by
sonication for 5 mins and assayed for drug content by developed HPLC method.
Percent Drug loading (DL%) was calculated using the equation:
DL (%) = WDL/WNP×100 ………………………………………….. (7)
where WDL = weight of Drug in Np and WNP = weight of Np
Poly (ethylene sebacate) Nanoparticles of Anti HIV-Anti tubercular drug combinations
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d) Zeta Potential
Zeta potential of nanoparticle dispersion was measured using Malvern Zetasizer
Nanoseries using DTS Nano software. Nanoparticle dispersion was centrifuged at
15,000 rpm for 30 min at 20ºC. The resultant pellet was washed and redispersed with
distilled deionized water (nanoparticles 100µg/ml) by sonication. Samples were filled
in to the folded capillary cell and zeta potential was measured. Each sample was
analyzed in triplicate.
e) Scanning Electron Microscopy (SEM)
The morphology/shape of nanoparticles was determined by SEM (JSM-6380-LA,
JEOL, Tokyo, Japan). A drop of colloidal dispersion was deposited onto a carbon tape
and dried under vacuum. The samples were sputtered with platinum using an auto fine
coater prior to analysis (JFC-1600, JEOL, Tokyo, Japan).
f) Fourier Transform Infrared (FTIR) Spectroscopy
FT-IR for rifampicin, lopinavir, PES, PLGA, PLA, the nanoparticles and excipients
were recorded on a Perkin-Elmer FTIR spectrophotometer by the KBr disk method
from 4000 to 500 cm-1. Samples were crushed to a fine powder, mulled with
anhydrous potassium bromide, compressed to form a thin transparent pellet and
subjected to FTIR.
g) Differential Scanning Calorimetry (DSC)
DSC thermograms of rifampicin, lopinavir, PES, PLGA, PLA, the nanoparticles and
excipients were recorded on a Perkin Elmer Pyris 6 DSC (PerkinElmer, Netherlands)
system in the temperature range 40 -300°C at a heating rate of 10°C /min in a
dynamic nitrogen atmosphere (20 mL/min). A sample of 5-6 mg was sealed in an
aluminum pan and an empty sealed aluminum pan was used as the reference.
h) Powder X-Ray Diffraction (PXRD)
Powder XRD patterns were obtained for rifampicin, lopinavir, PES, PLGA, PLA and
the nanoparticles were recorded using a Rigaku Miniflex diffractometer, with Cu Kα
target tube, NaI detector, variable slits, a 0.050 step size, operated at a voltage of 30
kV, 15mA current, at 2θ/min scanning speed, and scanning angles ranged from 8-
60º(2θ).
i) Hydrophobicity evaluation of nanoparticles (contact angle measurement)
Hydrophobicity of rifampicin, lopinavir, PES, PLGA, PLA and the nanoparticles was
evaluated by measuring the static contact angle of nanoparticles/drug pellet. Briefly,
nanoparticle pellets were prepared in KBr press using 25 mg freeze dried
Poly (ethylene sebacate) Nanoparticles of Anti HIV-Anti tubercular drug combinations
123 Particulate Carriers as Drug Delivery Systems for Anti-Tubercular and Anti-Cancer Agents
nanoparticles and press it at 10 tone pressure for 1 minute. Drop water contact angles
were measured (Kruss contact angle measuring instrument G10, Germany) using
approximately 5µl drop of Milli-Q water. Results are presented as an average of 3
measurements.
6.4.4. Stability study
The nanoparticles were freeze dried packed and sealed in amber glass vials and
subjected to stability studies as per the ICH guidelines at 300C/65RH and 400C/75RH.
6.4.5. Pharmacokinetic and biodistribution of nanoparticles
a) Pharmacokinetic study
Male Wistar rats (200-250gms) (n=6) were fasted 12-18h prior to dosing. Rats were
divided into four groups, one group received plain drug dispersion, the second group
received RIF-LOPI PES Np’s, third group received RIF-LOPI PLGA Np’s and fourth
group received RIF-LOPI PLA Np’s (equivalent to 10mg/kg body weight of each RIF
and LOPI) administered by oral gavage. Blood samples were withdrawn from the
retro-orbital plexus prior to dosing and at 1, 2, 4, 6, 12 and 24h post dosing and
collected in tubes containing 4.1% EDTA. Plasma was separated and extracted and
evaluated for drug content by HPLC. The peak plasma concentration (Cmax) and
peak plasma time (Tmax) were obtained by visual data inspection. The area under
plasma drug concentration over time curve (AUC0–t) and t1/2 were calculated using
BASICA software.
b) Biodistribution study
Rats used in the pharmacokinetic study were euthanized at 24h by excessive carbon
dioxide, lungs, liver, spleen and kidney were isolated, placed in phosphate buffered
saline (PBS) pH 7.4 and homogenized using a tissue homogenizer. RIF and LOPI was
extracted and evaluated for drug content by HPLC.
All experimental procedures were reviewed and approved by the Institutional Animal
Ethics Committee (IAEC) of Institute of Chemical Technology, Department of
Pharmaceutical Sciences and Technology, Mumbai, India (ICT/IAEC/2011/P71).
6.5. RESULTS AND DISCUSSION
6.5.1. Preparation and optimization of nanoparticles
Emulsion solvent evaporation method was selected for the preparation of RIF-LOPI
loaded PES nanoparticles. Method employs a solution of drug(s) and polymer in a
water immiscible solvent is emulsified in water with the aid of surfactant/emulsifying
agent. Method is advantageous for good entrapment efficiency for combination of
Poly (ethylene sebacate) Nanoparticles of Anti HIV-Anti tubercular drug combinations
124 Particulate Carriers as Drug Delivery Systems for Anti-Tubercular and Anti-Cancer Agents
hydrophobic agents (Jeffery et al., 1991, Okochi et al., 2000) compared to
nanoprecipitation method. In nanoprecipitation method when combinations of two
drugs are used, one drug which has more affinity towards organic phase displaces the
other drugs in an aqueous phase, whereas due to water immiscibility this phenomenon
is very low with emulsion solvent evaporation method.
For obtaining nanoparticles, studies were started with a prototype formula consisting
of the 100mg PES, 20mL aqueous phase, 10mL organic phase (dichloromethane),
25mg RIF, 25mg LOPI and 50mg PVA. The parameters were varied with this
prototype formula are as below
a) Effect of PVA concentration
Table 6.10 : Effect of PVA concentration on %EE and particle size
RLPES/1 RLPES/2 RLPES/3 Particle size (nm) 401.0 ± 25 302.5 ± 30 243.9 ± 22
PI 0.246 ± 0.103 0.293 ± 0.098 0.302 ± 0.102 %EE RIF 28.22 ± 1.67 % 27.37 ± 1.13 23.75 ± 3.04
% EE LOPI 93.7 ± 0.98 93.36 ± 1.01 92.76 ± 1.14
Figure 6.5: Effect of RIF-LOPI:PVA ratio on %EE and particle size
As seen from figure 6.5 while PVA concentration did not significantly influence
entrapment efficiency (P>0.05). A significant reduction in particle size (P<0.05) was
seen with increase in PVA concentration (table 6.10). PVA exerts its stabilizing effect
by adsorbing at the droplet interface thus reducing surface tension and promoting
mechanical and steric stabilization. Similar data is reported in literature for other
drugs (Lamprecht et al., 2001, Patil et al., 2008).
Poly (ethylene sebacate) Nanoparticles of Anti HIV-Anti tubercular drug combinations
125 Particulate Carriers as Drug Delivery Systems for Anti-Tubercular and Anti-Cancer Agents
b) Effect of rifampicin to lopinavir ratio
Figure 6.6: Effect of RIF to LOPI ratio on %EE and particle size
As shown in figure 6.6 RIF:LOPI ratio did not significantly influence particle size
(P>0.05). RIF showed maximum entrapment efficiency of ~60% when no LOPI
added during the preparation of nanoparticles (table 6.11). Addition of LOPI together
with RIF significantly (P<0.05) decrease the entrapment efficiency of RIF while
entrapment efficiency of LOPI was not affected. LOPI and RIF compete for the
organic phase however being hydrophobic LOPI (aqueous solubility 0.0019g/L)
preferably dissolves in the organic phase, as a consequence RIF (aqueous solubility
1.4g/L) partitions in to the aqueous phase. This is observed as a decrease in
entrapment efficiency of RIF with increase in LOPI concentration.
Table 6.11: Effect of RIF to LOPI ratio on %EE and particle size
RLPES/4 RLPES/5 RLPES/6 RLPES/2 Particle size
(nm) 299.3 ± 20 283.1 ± 25 273.6 ± 28 302.5 ± 30
PI 0.311 ± 0.088 0.362 ± 0.091 0.336 ± 0.110 0.293 ± 0.098 %EE RIF 60.15 ± 2.27 49.81 ± 1.08 47.24 ± 1.19 27.37 ± 1.13
% EE LOPI - 91.84 ± 2.01 92.12 ± 1.15 93.36 ± 1.01
c) Effect of drug: polymer ratio
Effect of different drug:PES ratio (RIF:PES-1:0.53) on entrapment efficiency and
particle size is shown in figure 6.7. Varying the drug:PES ratio from 1:1 to 1:3 did not
significantly (P>0.05) affect entrapment efficiency of RIF and LOPI (table 6.12). A
significant (P<0.05) increase in particle size was observed with increase in drug:PES
ratio from 1:1 to 1:3. The increase in particle size with increasing polymer
concentration was discussed in section 5.1.1.
Poly (ethylene sebacate) Nanoparticles of Anti HIV-Anti tubercular drug combinations
126 Particulate Carriers as Drug Delivery Systems for Anti-Tubercular and Anti-Cancer Agents
Table 6.12: Effect of drug to polymer ratio on %EE and particle size
RLPES/8 RLPES/9 RLPES/7 RLPES/10 Particle size
(nm) 239.9 ± 15 258.4 ± 18 287.6 ± 8.27 319.89 ± 9
PI 0.322 ± 0.108 0.278 ± 0.10 0.297 ± 0.104 0.390 ± 0.098 %EE RIF 39.03 ± 1.98 41.71 ± 2.15 48.1 ± 2.07 45.73 ± 1.13
% EE LOPI 94.93 ± 2.62 94.65 ± 2.35 95.3 ± 0.81 96.52 ± 2.11
Figure 6.7: Effect of Drug to Polymer ratio on %EE and particle size
Table 6.13: Optimized batch for RIF-LOPI PES, PLGA and PLA nanoparticles
RLPES/7 RIF:LOPI PES Np’s
RLPLGA/1 RIF:LOPI
PLGA Np’s
RLPLA/1 RIF:LOPI PLA Np’s
Average particle size 287.6 ± 8.27 nm 274.2 ± 14.9 nm 288.4 ± 13.2 nm %EE For RIF 48.1 ± 2.07 % 58.06 ± 0.78 % 58.07 ± 0.12 % %EE For LOPI 95.3 ± 0.81 % 95.1 ± 0.8 % 93.95 ± 0.22 % Drug Entrapped For RIF 16.63 ± 0.63 mg 17.89 ± 0.07 mg 18.0 ± 0.04 mg For LOPI 16.67 ± 0.14 mg 18.02 ± 0.08 mg 17.85 ± 0.04 mg RIF:LOPI ratio ~1:1 ~1:0.99 ~1:1.008 Total drug loading 24.98 ± 1.3 % 26.47 ± 1.2 % 26.2 ± 0.9 %
For comparative evaluation in vivo RIF-LOPI nanoparticles were also prepared using
FDA approved polymer PLGA (intermediate hydrophobicity) and PLA
(hydrophobic). PES was replaced with PLGA and PLA in RLPES/7 to get RIF-LOPI
loaded PLGA (RLPLGA/1) and PLA (RLPLA/1) nanoparticles respectively. Table
6.13 shows optimized batch for RIF-LOPI loaded PES, PLGA and PLA nanoparticles.
Despite the change in polymer we obtained comparable drug entrapment and
Poly (ethylene sebacate) Nanoparticles of Anti HIV-Anti tubercular drug combinations
127 Particulate Carriers as Drug Delivery Systems for Anti-Tubercular and Anti-Cancer Agents
RIF:LOPI ratio ~1:1 as seen in table 5.1. Average particle size was comparable with
the RIF-LOPI PES nanoparticles (RLPES/7).
6.5.2. Freeze-drying of nanoparticles
Freeze-drying of RIF-LOPI loaded PES, PLGA and PLA nanoparticle batches were
carried out using trehalose (10:1 by weight of nanoparticles) and lutrol-F-68 (0.1:1 by
weight of nanoparticles) revealed the best cryo-protection with a Sf/Si (Sf- final
particle size after freeze thaw, Si- initial particle size) ratio of <1.3 as optimized in
section 5.2.2. The data is shown in table 6.14.
Table 6.14: Freeze drying of RIF-LOPI loaded PES, PLGA and PLA nanoparticle
Sr. No Freeze dried NPDDS Size before
FD (Si) Size after FD (Sf)
Sf/Si ratio
1 RIF-LOPI PES Np (RLPES/7) 289.1 335.9 1.16 2 RIF-LOPI PLGA Np (RLPLGA/1) 279.5 332.6 1.19 3 RIF-LOPI PLA Np (RLPLA/1) 297.2 350.7 1.18
6.5.3. Evaluation and physical characterization
a) Zeta Potential
Nanoparticles exhibited a negative zeta potential due to free terminal hydroxyl group
of PES and carboxylic group of PLGA and PLA (table 6.15). Zeta potential values
ranged from -25 to – 35 mV which is an indicator of good colloidal stability (Sugrueet
al., 1992).
Table 6.15: Zeta potential of RIF-LOPI loaded PES, PLGA and PLA nanoparticle
NPDDS Zeta Potential mV RIF-LOPI PES Np (RLPES/7) -21.2 ± 3.4 mV RIF-LOPI PLGA Np (RLPLGA/1) -19.5 ± 2.9 mV RIF-LOPI PLA Np (RLPLA/1) -24.1 ± 4.1 mV
b) Scanning Electron Microscopy (SEM)
a) b) c)
Figure 6.8: SEM images of a) RIF-LOPI PES Nps b) RIF-LOPI PLGA Nps c) RIF-LOPI PLA Nps
Scanning Electron Microscopy (SEM) revealed polydispersed nanoparticles with
spherical morphology (figure 6.8).
Poly (ethylene sebacate) Nanoparticles of Anti HIV-Anti tubercular drug combinations
128 Particulate Carriers as Drug Delivery Systems for Anti-Tubercular and Anti-Cancer Agents
c) Fourier Transform Infrared (FTIR) Spectroscopy
FTIR studies are rapid method of accessing drug: excipients interaction in the
formulation. FTIR of RIF, LOPI, PES, PLGA, PLA and their respective nanoparticles
along with excipients revealed all the characteristic peaks of RIF and LOPI. RIF has
principal peaks at wavenumbers 1250, 1567, 976, 1098, 1064, 1650 cm−1which are
also found in RIF PES, PLGA and PLA nanoparticles. LOPI has principal peaks at
wavenumbers 1090, 1061, 1142, 987, 1050, 1009 cm−1. No drug: excipient
interaction was evident in the spectra (figure 6.9).
Rifampicin Lopinavir
Poly vinyl alcohol Poly (ethylene sebacate)
PLGA PLA
Poly (ethylene sebacate) Nanoparticles of Anti HIV-Anti tubercular drug combinations
129 Particulate Carriers as Drug Delivery Systems for Anti-Tubercular and Anti-Cancer Agents
RIF-LOPI PES Nps RIF-LOPI PLGA Nps
RIF-LOPI PLA Nps
Figure 6.9: FTIR of drugs, excipients and nanoparticles
d) Differential Scanning Calorimetry (DSC)
a) b)
c)
Figure 6.10: DSC thermogram of a)RIF-LOPI PES Nps b) RIF-LOPI PLGA Nps c) RIF-LOPI PLA Nps
Poly (ethylene sebacate) Nanoparticles of Anti HIV-Anti tubercular drug combinations
130 Particulate Carriers as Drug Delivery Systems for Anti-Tubercular and Anti-Cancer Agents
DSC enables detection of all the processes in which energy is required or produced
(i.e. endothermic and exothermic phase transformations). The thermograms of RIF,
LOPI, PES, PLGA, PLA and their respective nanoparticles along with excipients are
shown in figure 6.10. Pure RIF and LOPI revealed a sharp melting endotherm
corresponding to their melting point indicating crystalline nature. The disappearance
of RIF and LOPI melting endotherm in their respective nanoparticle suggests
remarkable decrease in crystallinity.
e) Powder X-Ray Diffraction (PXRD)
Crystallinity in the sample is reflected by a characteristic fingerprint region in the
diffraction pattern. The XRD spectra of RIF, LOPI, PES, PLGA, PLA and their
nanoparticles are shown in figure 6.11. RIF, LOPI, PES, PLGA and PLA are highly
crystalline powders showing characteristic sharp diffraction peaks. These sharp
diffraction peaks disappeared in the respective nanoparticles indicating
amorphization/remarkable decrease in crystallinity of the drug.
Rifampicin Lopinavir
Poly (ethylene sebacate) PLGA
PLA RIF-LOPI PES Nps
RIF-LOPI PLGA Nps RIF-LOPI PLA Nps
Figure 6.11: pXRD spectra of RIF, LOPI, PES, PLGA, PLA and their respective nanoparticles
Poly (ethylene sebacate) Nanoparticles of Anti HIV-Anti tubercular drug combinations
131 Particulate Carriers as Drug Delivery Systems for Anti-Tubercular and Anti-Cancer Agents
f) Hydrophobicity evaluation of nanoparticles (contact angle measurement)
As shown in table 6.16 hydrophobicity in term of contact angle of lopinavir was high
(91.45 ± 1.44) followed by rifampicin (74.33 ± 1.15), PLA (71.46 ± 1.47), PLGA
(61.1 ± 2.81) and PES (60.0±1.0). Hydrophobicity of LOPI and RIF was significantly
decreased when formulated in polymeric nanoparticles. PLA showing higher contact
angle was relatively more hydrophobic than PES and PLGA. PES and PLGA
exhibited comparable hydrophobicity.
Table 6.16: Hydrophobicity in terms of water contact angle of nanoparticles
Sr. No. Formulations Contact angle (n=3) 1. Rifampicin 74.33±1.15 2. Lopinavir 91.45±1.44 2. PES 60.0 ± 1 3. PLGA 61.1 ± 2.81 4. PLA 71.46 ± 1.47 5. RIF-LOPI PES Np (RLPES/7) 48.93 ± 2.03 4. RIF-LOPI PLGA Np (RLPLGA/1) 47.28 ± 1.32 7. RIF-LOPI PLA Np (RLPLA/1) 58.98 ± 2.57
6.5.4. Stability study
Freeze dried NPDDS were stable for 6 months at 30°C/60%RH and 40°C/75%RH.
All the nanoparticles revealed good redispersibility, no significant change in particle
size (as indicated by Sf/Si ratio<1.3) and drug content >90% suggest stability (table
6.17-6.19).
Table 6.17: Stability results of RLPES/7 at 300C/65%RH and 400C/75%RH
30°C/65%RH 40°C/75%RH
Drug content Sf/Si ratio
Drug content Sf/Si ratio RIF LOPI RIF LOPI
Initial 99.42 ±3.12
99.48 ±2.29 1.160 99.42
±3.12 99.48 ±2.29 1.160
1 month 97.72 ±1.46
98.85 ±2.65 1.171 98.14
±1.86 98.29 ±1.36 1.172
3 month 97.45 ±1.92
97.96 ±1.48 1.169 97.87
±2.01 97.54 ±1.93 1.171
6 month 96.99 ±1.28
97.23 ±2.02 1.178 97.12
±1.37 97.44 ±1.26 1.181
Poly (ethylene sebacate) Nanoparticles of Anti HIV-Anti tubercular drug combinations
132 Particulate Carriers as Drug Delivery Systems for Anti-Tubercular and Anti-Cancer Agents
Table 6.18: Stability results of RLPLGA/1 at 300C/65%RH and 400C/75%RH
30°C/65%RH 40°C/75%RH Drug content Sf/Si
ratio Drug content Sf/Si
ratio RIF LOPI RIF LOPI
Initial 98.99 ±2.49
99.95 ±3.05 1.190 98.99
±2.49 99.95 ±3.05 1.190
1 month 99.12 ±2.92
98.92 ±2.27 1.187 99.04
±2.12 99.19 ±2.33 1.192
3 month 98.59 ±1.78
98.63 ±1.86 1.191 98.95
±2.10 98.43 ±1.98 1.190
6 month 97.43 ±1.89
97.90 ±1.02 1.195 96.42
±1.94 97.04 ±1.79 1.197
Table 6.19: Stability results of RLPLA/1 at 300C/65%RH and 400C/75%RH
30°C/65%RH 40°C/75%RH Drug content Sf/Si
ratio Drug content Sf/Si
ratio RIF LOPI RIF LOPI
Initial 99.69 ±2.82
100.5 ±3.25 1.180 99.69
±2.82 100.5 ±3.25 1.180
1 month 99.66 ±2.22
99.59 ±2.93 1.178 99.79
±2.36 99.84 ±2.23 1.175
3 month 98.25 ±1.03
98.43 ±1.39 1.179 98.36
±2.25 98.84 ±1.35 1.181
6 month 97.66 ±1.90
97.41 ±1.87 1.181 97.93
±1.83 97.32 ±1.74 1.187
6.5.5. Pharmacokinetic and biodistribution of nanoparticles
Figure 6.12: Pharmacokinetic profile of RIF-LOPI nanoparticles
Pharmacokinetic evaluation of RIF-LOPI nanoparticles revealed significantly higher
and sustained plasma drug concentration, delayed Tmax and enhanced oral
bioavailability for both RIF and LOPI compared to plain drugs (figure 6.12). T1/2
values were significantly higher with the nanoparticles for both RIF and LOPI (table
Poly (ethylene sebacate) Nanoparticles of Anti HIV-Anti tubercular drug combinations
133 Particulate Carriers as Drug Delivery Systems for Anti-Tubercular and Anti-Cancer Agents
6.20). Relative oral bioavailability increased upto ~150% for RIF and ~200% for
LOPI with RIF-LOPI PES NPs and RIF-LOPI PLGA NPs respectively. RIF-LOPI
PLA NPs revealed even greater increase in relative oral bioavailability of ~200% for
RIF and ~255% for LOPI compared with plain drug. Hydrophobic particles are
readily taken up M-cells of by Peyer’s patches and lymphoid tissues. The contact
angle value intable 6.15 revealed PLA as the most hydrophobic polymer while PES
and PLGA exhibit comparable hydrophobicity. The hydrophobic nature enables
higher uptake through the Peyer’s patches and lymphoid tissues in the GI tract thereby
exhibiting enhanced bioavailability.
Table 6.20: Pharmacokinetic parameters of RIF-LOPI nanoparticles
PK Parameters
RIF-LOPI Dispersion RLPES/7 RLPLGA/1 RLPLA/1
RIF LOPI RIF LOPI RIF LOPI RIF LOPI
C max (μg/ml) 6.42 ±0.85
2.05 ±0.23
7.47 ±1.64
4.20 ±0.3
8.07 ±0.67
4.78 ±0.32
10.26 ±0.83
6.22 ±0.37
T max (h) 1 2 4.4 ± 0.89 4 4 4 6 6
Slope -0.014 ±0.002
-0.018 ±0.005
-0.023 ±0.003
-0.026 ±0.005
-0.021 ±0.001
-0.025 ±0.001
-0.028 ±0.001
-0.035 ±0.001
Kel(h-1) 0.034 ±0.005
0.042 ±0.012
0.053 ±0.008
0.061 ±0.013
0.048 ±0.003
0.058 ±0.004
0.065 ±0.004
0.081 ±0.003
T½(h) 6.68 ±2.89
7.33 ±4.02
13.23 ±2.09
11.64 ±2.06
14.25 ±1.11
11.91 ±0.81
10.62 ±0.74
8.51 ±0.35
AUC(μg/ml*h) 60.65 ±3.87
24.23 ±0.94
90.37 ±6.62
45.73 ±1.27
93.91 ±3.73
50.49 ±1.89
122.95 ±4.87
61.87 ±1.86
AUC infinity (μg/ml*h)
105.2 ±5.28
39.06 ±5.72
132.92 ±16.64
62.91 ±5.43
142.67 ±4.91
69.97 ±2.41
167.09 ±4.68
76.65 ±1.74
Bioavailability enhancement Ref. Ref. 149% 189% 155% 209% 203% 255%
Figure 6.13: Biodistribution profile of RIF-LOPI nanoparticles
Poly (ethylene sebacate) Nanoparticles of Anti HIV-Anti tubercular drug combinations
134 Particulate Carriers as Drug Delivery Systems for Anti-Tubercular and Anti-Cancer Agents
Following oral administration of RIF-LOPI PES and PLGA nanoparticles revealed
significantly higher lung concentration compared to RIF-LOPI solution. However, RIF-
LOPI PLA nanoparticles revealed maximum lung uptake (figure 6.13). In other organs
(liver, kidney and spleen) no significant difference were seen with the nanoparticles.
Enhanced bioavailability and lung concentration is attributed to rapid and high uptake via
M cells of the Peyer’s patches. High bioavailability of LOPI confirmed that entrapping
the drug in a nanoparticulate carrier could provide adequate protection from cytochrome
P450 even in the presence of a drug like RIF a known inducer of cytochrome P450 to
enable significantly enhanced bioavailability. This protective effect of the nanoparticles
could have enabled high bioavailability of LOPI despite being in combination with RIF
an inducer of cytochrome P450. Similar observation on the metabolism of PLA
nanoparticles of isoniazid in vivo is demonstrated (Zhou et al., 2005).
In conclusion, design of RIF-LOPI nanoparticles provides a promising approach for
combining the two drugs for improved therapy of tuberculosis in HIV patients.
6.5.6. Highlights
Emulsion solvent evaporation technique represents a simple approach for
simultaneous entrapment of two drugs in a ratio 1:1 with high drug loading
RIF-LOPI PLA nanoparticles revealed enhancement in bioavailability 205%
for RIF and 255% for LOPI
The high bioavailability of LOPI observed confirm the entrapping is a suitable
strategy of bioenhancement despite combination with RIF an inducer of
cytochrome P450.