International Journal of Pharmaceutics 461 (2014) 223–233
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
Intranasal delivery of streptomycin sulfate (STRS) loaded solid lipid nanoparticles to brain and blood Manoj Kumar a , Vandita Kakkar a , Anil Kumar Mishra b , Krishna Chuttani b , Indu Pal Kaur a,∗ a b
University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh 160014, India Institute of Nuclear Medicine and Allied Science (INMAS), Timarpur, New Delhi, India
a r t i c l e
i n f o
Article history: Received 8 August 2013 Received in revised form 8 November 2013 Accepted 18 November 2013 Available online 25 November 2013 Keywords: Streptomycin Intranasal Solid lipid nanoparticles Biodistribution Blood
a b s t r a c t Factors like unreliable and poor oral absorption, including an active Pgp-efflux point towards a compromised oral bioavailability (BA) of streptomycin sulfate (STRS). Latter instigates its parenteral use (i.m.) only. Furthermore, its chronic use leads to serious side effects like nephrotoxicity and ototoxicity. In the present study, we propose to develop streptomycin sulfate (STRS) loaded solid lipid nanoparticles (STRS-SLNs) for non-invasive intranasal (IN) delivery. STRS-SLNs were prepared using patented nanocolloidal aqueous dispersion technique (Indian Patent application 3093/DEL/2012). Small particle size (140.1 ± 7.0 nm) and significant entrapment efficiency (54.83 ± 2.1%) was achieved. Biodistribution studies using 99m Tc showed a 3.15 and 11.0 times higher concentrations in the brain and blood of mice, respectively, on IN administration of STRS-SLNs in comparison to free (F)-STRS. Lower concentrations (3.3 times) in kidneys implicate lower nephrotoxicity. Similarly a 12 and 4 times lower levels of drug in liver and spleen, respectively upon administration of STRS-SLNs as compared to F-STRS also indicate its lesser accumulation in these reticuloendothelial system organs. Lipophillic enclosure imparted to STRS, coupled with small particle size, and its purported ability to inhibit Pgp-efflux due to the presence of tween 80, is considered to be responsible for a better BA shown by STRS upon incorporation into SLNs. This is predicted to result in an effective treatment of all types of tuberculosis including cerebral tuberculosis as indicated by high relative distribution to brain in comparison to free-STRS. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Streptomycin (STR), an aminoglycoside, is a bactericidal antibiotic active against both gram-negative and gram-positive organisms, and Mycobacterium. It acts primarily by inhibition of protein synthesis leading to codon misreading and ultimately death of microbial cells (Chambers, 2005). Streptomycin sulfate (STRS) is indicated as a first line (though parenteral) antitubercular drug (ATD) and is especially suggested for (i) patients with hepatitis and other liver disorders induced by other hepatotoxic oral ATDs, viz. rifampicin, isoniazid and pyrazinamide. A non-hepatotoxic regimen consisting of streptomycin, ethambutol and a fluoroquinolone is recommended in such situations, for 18–24 months with or without 1 or 2 hepatotoxic ATDs; (ii) tuberculous meningitis, where ethambutol is suggested to be replaced by streptomycin; and, (iii) patients returning after default or relapse and now receiving the
∗ Corresponding author at: Department of Pharmaceutics, University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh-160014, India. Tel.: +91 172 2534191; fax: +91 172 2543101. E-mail address: [email protected] (I.P. Kaur). 0378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2013.11.038
8-month retreatment regimen, with an initial 2 months of intensive treatment with all first line drugs including streptomycin (WHO, 2010). Streptomycin suffers from serious risk of nephrotoxic reactions in patients with impaired kidney function; irreversible ototoxicity is also reported at high doses. Intranasal (IN) delivery is considered a promising alternative to intravenous route for a rapid delivery of therapeutic agents to the blood and brain for treating CNS disorders (Costantino et al., 2007; Hanson and Frey, 2008; Grassin-Delyle et al., 2012). It has been demonstrated that part of the therapeutic administered via the intranasal route may be delivered directly to the CNS along both the olfactory and trigeminal nerve (Thorne et al., 2008). IN route provides advantages including a large surface area for absorption, rapid achievement of target drug levels, avoidance of first pass metabolism and improvement of drug bioavailability (BA). Furthermore, this delivery route, in comparison to the parenteral route shows maximal patient comfort and compliance (Dragphia et al., 1995; Illum, 2000, 2003; Wermling et al., 2001; Dorman et al., 2002; Vyas et al., 2005). However, the rapid removal of formulation by mucociliary clearance often limits the BA of drugs delivered intranasally. Therefore, retaining drug longer in the nasal cavity using options like mucoadhesives or nanoparticulate
224
M. Kumar et al. / International Journal of Pharmaceutics 461 (2014) 223–233
systems can help maximize the utility of IN delivery. In addition to the prolonged drug residence time, for poorly absorbed drugs such as streptomycin, nanoparticulate systems can help to enhance the permeability across the epithelial membrane. Latter will be essential for achieving improved systemic delivery (Garcia-Fuentes et al., 2005; Müller et al., 2006). Solid lipid nanoparticles (SLNs) are drug carriers with a particle size 50% drug within the SLN/NCD system, considering the highly hydrophilic nature of STRS as indicated by its very high negative log p of −6.7. Though hydrophilic drugs are proposed to be incorporated into SLNs but achieving significant payload and entrapment efficiency into the lipophilic carrier system is an issue (Kisel et al., 2001; Bhandari and Kaur, 2013). Novelty of the developed NCD lies both in the method of preparation and in the fact that more concentrated nanoparticles/volume are achieved by the present method, in comparison to the corresponding SLN dispersions prepared using a generally employed microemulsion method. Latter involves a 1:10–1:100 dilution of the microemulsion with cold water (Gasco, 1993, 1997; Gohla and Dingler, 2001). Such dilute dispersions are recommended to be lyophilized and reconstituted before use (Gasco, 1993, 1997). Latter will invariably result in aggregates and hence some microsize particles rather than uniformly distributed nanostructures. Another process recommended for concentrating the dilute nanodispersions is diafiltration. Personally speaking, we have a strong reservation to the later method as it is in contrast to the very definition of SLNs which states that they constitute ‘lipid nanoparticles dispersed in an aqueous surfactant solution’. Upon diafiltration the surfactant present in the aqueous dispersion is removed and the remaining nanocolloidal dispersion will again tend to aggregate and will be destabilized. Presence of surfactant molecules in the aqueous medium is very critical to the stability of the nanosystem and are specific to that system. Hence, it is highlighted here that the SLN/NCD system presently reported for the encapsulation of STRS is unique in the sense that inspite of a high solid content (8.8% which is 10 to 100 times higher than normally reported system) the developed system was very stable showing no significant change in size after storage of up to 2 years under refrigeration.
Fig. 1. TEM micrograph of STRS-SLNs.
Latter is attributable to maintaining the innate nature in which the system was developed. Furthermore the amount of drug which can be loaded/volume in the present system also tends to be high (due to lower dilution) and it is thus practical to administer clinically the otherwise high dose of such agents. 6.1. Particle size analysis Average particle size of STRS-SLNs was 140.1 ± 7.0 nm with a polydispersity index (P.I.) of 0.281, when measured using photon correlation spectroscopy (Malvern, Zetasizer UK). A small PI indicates uniformly produced nanodispersion; no micronized particles were observed in the entire population. The small particle size achieved with SLNs and encapsulation of STRS within lipidic core is expected to facilitate transport of drug across the nasal mucosal barriers both into the cerebral tissues and systemic circulation (Choi and Maysinger, 2013). 6.2. TEM TEM analysis revealed spherical particles in size range of the order 90–120 nm. The size of the nanoparticles observed under TEM (Fig. 1) was smaller than the results obtained using Mastersizer® . This may however be attributed to the fact, that Mastersizer® is based on the principle of dynamic light scattering, which at times may not detect small particles, due to the higher brownian movement of these particles. TEM on the other hand, involves observation of a fairly small number of representative particles (in 2–3 fields) of a dilute dispersion such that the particle size observed under TEM do not match with results obtained by the PCS technology which gives population statistics. 6.3. Total drug content (TDC) and entrapment efficiency (EE) TDC of STRS-SLNs was estimated to be 88.23 ± 1.2% (n = 6) and the EE of STRS-SLNs was 54.83 ± 2.1% (n = 6). Entrapment values of >50% for STRS which is a highly hydrophilic molecule (log p = −6.7) have never been reported earlier in the available literature. Pandey and Khuller (2007) have worked extensively on several antitubercular drugs including streptomycin. They report polymeric nanoparticles of STRS with an EE of 32%; particle size of the formulation prepared by them was 153 nm. Compritol® 888
M. Kumar et al. / International Journal of Pharmaceutics 461 (2014) 223–233
227
Fig. 2. DSC of a) STRS b) Compritol® 888 ATO c) STRS-SLNs.
ATO was presently chosen as the lipid component because it tends to result in dispersions with small particle size and also shows good distribution into brain (Cavalli et al., 1997; Kaur et al., 2008). Furthermore, the surfactant to co-surfactant, ratio was kept suitably high to achieve not only a small size but also a significant EE with a better brain distribution (Kakkar and Kaur, 2011; Kuo and Chung, 2011; Bhandari and Kaur, 2013; Kakkar et al., 2013b; Singh et al., 2013). 6.4. DSC DSC is a thermoanalytical technique in which the difference in the amount of heat required to maintain the sample and reference at the same temperature is measured as a function of temperature and time. The breakdown or fusion of the crystal lattice by heating or cooling the sample yields information about the internal polymorphism, crystal order, or glass transition processes (Uner, 2006). It uses the fact that different lipid modifications possess different melting points and enthalpies. The basic principle underlying this technique is that when the sample undergoes a physical
transformation (such as melting, desolvation), some amount of heat is required to flow to it depending on whether the process is exothermic or endothermic to maintain both the reference and sample at the same temperature. DSC measures this heat flow into or from the sample when it is heated or cooled and thus provides qualitative and quantitative information about physicochemical changes (i.e. endothermic or exothermic nature of the process or changes in heat capacity). In case of pure streptomycin sulfate, melting endotherm appeared at 151.81 ◦ C corresponding to its melting point and was associated with 252.3 J/g of enthalpy, while Compritol® 888 ATO showed a sharp peak at 74.72 ◦ C and an enthalpy of 122.6 J/g. STRSSLNs showed an endothermic shift to 98.77 ◦ C and a significantly lowered heat flow of 66.79 J/g (Fig. 2) indicating incorporation of drug into the lipid matrix. A significant lowering of enthalpy for STRS-SLNs versus pure lipid indicates a reduction in particle size and the change in polymorphic state of the lipid from its crystalline  form to the amorphous (␣,  ) form (Müller et al., 2006) with more imperfections in the crystal lattice. Latter results in comfortable incorporation of more amount of drug within the lipid molecules.
228
M. Kumar et al. / International Journal of Pharmaceutics 461 (2014) 223–233 Counts
Table 1 Peaks of FTIR spectra for STRS, Compritol® 888 ATO and STRS-SLNs.
400
200
0
10
Counts
20
30
40
Position [°2Theta] (Copper (Cu))
a) 6000
4000
2000
0
10
20
30
40
Position [°2Theta] (Copper (Cu))
b)
Sr no
STRS (cm−1 )
Compritol® 888 ATO (cm−1 )
STRS-SLNs (cm−1 )
1. 2. 3. 4. 5. 6 7. 8. 9. 10. 11.
3364.7 2933.5 1671.5 1460.5 1124.9 1052.9 862.6 618.5
3348.9 2919.5 1739.9 1624.9 1468.4 1382.1 1174.8 11097 1054.6 722.1 549.7
3432.3 2921.1 1736.8 1651.3 1463.2 1352.8 1251.3 1105.8 9506 838.7 538.5
3364.7 cm−1 (N–H stretching) in STRS spectra, 3448.9 cm−1 (due to –OH– stretch of –COOH group) in Compritol® 888 ATO and a similar stretch is observed in STRS-SLNs at 3432.3 cm−1 . It may however be noted that the peak due to –OH stretch is reported to be broad (as also observed by us, Fig. 4b), while the peak observed for prepared SLNs at 3432.3 cm−1 tends to be sharper.
Counts
6.7. In vitro release
1000
500
0
10
20
30
40
Position [°2Theta] (Copper (Cu))
c) Counts 600
400 200
0
10
d)
20
30
40
Position [°2Theta] (Copper (Cu))
Fig. 3. P-XRD of a) STRS b) Compritol® 888 ATO c) blank SLNs d) STRS-SLNs.
The drug release from STRS-SLNs at 37 ± 0.5 ◦ C is shown in Fig. 5. A 100% release of STRS from STRS-SLNs was achieved at the end of 24 h, while in case of the F-STRS complete release was observed within 2 h. The in vitro drug release from STRS-SLNs showed a phasic behavior. At initial time points of up to 1 h a superclass II release was observed. This may be ascribed to either a fast release of the free unentrapped drug (%EE of STRS-SLNs was 54.85% and the remaining 45.17% of free-STRS is also present in the SLN dispersion) across the dialysis membrane or burst release and/or erosion of the drug from the outer phospholipid/surfactant layer of SLNs as is expected for a hydrophilic drug (Bhandari and Kaur, 2013). Subsequent release (post 1 h), was non fickian i.e., a combination of diffusion and erosion. STRS is a highly water soluble drug and hence its adsorption on the surface of the lipidic nanoparticles and its consequent release into the dialysis bag coupled with the passage of very small SLNs across the dialysis bag may be responsible for the second fast release phase, starting from 1 h onwards till 8 h, where there is a hump (a further increase) up to 12 h. Of the various release models, Higuchi release model was best fitted for the developed solid lipid nanoparticles (Table 2) at all the time points.
6.5. Powder X-ray diffraction (PXRD)
6.8. Stability study
PXRD patterns of STRS, Compritol® 888 ATO, lyophilized blank SLNs (B-SLNs) and lyophilized STRS-SLNs are shown in Fig. 3. PXRD pattern of STRS shows an amorphous nature while the PXRD of Compritol® 888 ATO showed sharp peaks at 2 scattered angles 21.16, 23.37, 23.52 and 35.76, establishing its crystalline nature. However, absence of these characteristic peaks in lyophilized BSLNs and STRS-SLNs sample, confirm the loss of crystallinity of the lipid and its shift towards the amorphous state. Latter indicates probable incorporation of STRS into the lipid matrix of the SLNs in the final formulation.
After 2 years of storage at 5 ± 3 ◦ C, the STRS-SLN dispersion was found to be stable, with no significant increase in size and depicting 100% assay and entrapment efficiency (p < 0.001) with respect to initial values at the end of 2 years (Table 3). Part of this stability (as discussed earlier) may be attributed to the maintenance of SLNs in their native state in which they were produced comprising suitable concentrations of surfactant in the aqueous phase in which they are dispersed. Another reason may be attributed to the composition of SLNs including an appropriate surfactant: co-surfactant ratio and the method of preparation which results in a concentrated nanoparticle dispersion with high solid content.
6.6. FTIR spectroscopy The FTIR spectra for F-STRS, Compritol® 888 ATO and the developed STRS-SLNs are shown in Fig. 4. Closer inspection of various peaks (Table 1) obtained in these spectra indicate two new peaks at 1651.3 cm−1 (N–H bending) and at 1251.3 cm−1 (C–N stretching) in the STRS-SLNs spectra. These new peaks indicate probable incorporation of STRS in SLNs. Further to this a peak is observed at
6.9. In vivo biodistribution studies Biodistribution studies of STRS-SLNs and F-STRS in the brain and blood of mice following IN administration, were conducted using technetium labeled (99m Tc-labeled) free drug solution or its developed SLNs at different times post administration (Table 4). The
M. Kumar et al. / International Journal of Pharmaceutics 461 (2014) 223–233
229
Fig. 4. FTIR spectrum of a) STRS b) Compritol® 888 ATO c) STRS-SLNs.
results showed achievement of 3.15 and 11.0 times, higher radioactivity in brain and blood respectively when STRS-SLNs (Table 4) were administered IN, in contrast to F-STRS, thus confirming the efficacy of the developed system. There was 7.7 times and 10.31
times enhancement in AUC 0 − ∞ and AUMC 0 − ∞ for STRS-SLNs as compared to F-STRS in blood, and 3.5 times and 5.8 times in brain respectively. Further 2.55 times and 1.61 times enhanced mean residence time was observed for STRS-SLNs as compared to F-STRS
230
M. Kumar et al. / International Journal of Pharmaceutics 461 (2014) 223–233
Table 2 Invitro release kinetics. R2 in different release models
Time points (h)
Value of n
Mechanism of drug release
Korsmeyer peppas
Higuchi
First order
Zero order
0.25 0.5 1 2 4 8 12 24
n = 0.93
Super class II, erosion
0.9374
0.9617
0.8163
0.9187
n = 0.58
Non fickian diffusion and erosion controlled rate release
0.9994
0.9995
0.9736
0.9986
0.8591
0.8618
0.7568
0.8162
Table 3 Stability study parameters during storage of STRS-SLNs at 5 ± 3 ◦ C (n = 6). Time points
Av. particle size (nm)
Total drug content* (%)
Entrapment efficiency* (%)
0 times 6 months 12 months 24 months
141.0 ± 5.0 105.0 ± 12.0 125.0 ± 8.0* 144.1 ± 6.0*
89.0 ± 2.5 88.6 ± 1.2* 88.2 ± 1.6* 89.92 ± 2.4*
54.83 ± 2.1* 53.14 ± 1.5* 53.87 ± 1.2* 52.07 ± 1.8*
*
*
* Statistically insignificant difference (p < 0.001) in average particle size at 0, 12 and 24 months of stability samples. Further, no statistically significant difference (p < 0.001) in total drug content and entrapment efficiency was observed at any time point.
Fig. 5. In vitro drug release of F-STRS and STR-SLNs in phosphate buffer (pH 7.4). ANOVA indicates that values at one time point are significantly different (p < 0.001) from the preceeding and succeeding time points except where marked suitably (*).
in blood and brain, respectively. A faster tmax (2 h) obtained with SLNs in comparison to free drug (4 h) in the brain of the mice are indicative of the rapid transport of STRS-SLNs to the brain post IN administration (Table 5). STRS-SLNs showed 3.3 times lower concentrations of drug in kidney as compared to the free drug (Table 4) post intranasal administration. Similarly 12.0 and 4.0
times lesser drug concentrations were observed in liver and spleen tissues, respectively (Fig. 6). This may also indirectly indicate low incidence of nanoparticulate related toxicity, higher systemic availability and prolonged residence time (due to reduced metabolic conversion). Intranasal administration of STRS-SLNs is purported to bypass the RES organs, as a result of which their systemic availability and therefore their concentration across the BBB is expected to be higher in comparison to the poorly available free-STRS. Former was supported by the results obtained in the biodistribution studies in liver and spleen (Fig. 6). Furthermore improved brain bioavailability may be assigned to the use of tween 80 in the preparation of SLNs which is reported to improve brain delivery of nanoparticles by (i) solublization of endothelial cell membrane lipids and membrane fluidization, (ii) through the temporary opening of inulin spaces (Zensi et al., 2009), (iii) endocytosis of nanoparticles, and (iv) inhibition of efflux system, especially P-gp present on the intranasal membrane and also the BBB (Wermling et al., 2001; Awasthi et al., 2005). Furthermore, the lipidic nature of SLNs may also help in their preferred passage across the lipoidal biological membranes while the phospholipid coat may have an added advantage in assisting in their movement via paracellular pathways (Barakat et al., 2006).
Fig. 6. % Radioactivity/gram tissue in liver and spleen post administration of free drug and STRS-SLNs.
M. Kumar et al. / International Journal of Pharmaceutics 461 (2014) 223–233
231
Table 4 Biodistribution of 99m Tc-F-STRS and 99m Tc-STRS-SLNs in blood, brain and kidney at different time intervals after intranasal (IN) administration in Balb/c mice. Concentration of STRS (% radioactivity/g at different times) and place this title above the various time points. Formulation and route of administration Intransal (IN)
Organ/tissue
0.25 h
0.5 h
1h
2h
4h
24 h
Free drug (F-STRS)
Blood Brain Blood Brain Brain Blood Kidney Kidney
0.017 ± 0.002 0.070 ± 0.006 0.120 ± 0.003 0.080 ± 0.003 1.142 ± 0.160 7.059 ± 0.530 0.20 ± 0.003 0.09 ± 0.002
0.014 ± 0.005 0.085 ± 0.003 0.150 ± 0.060 0.320 ± 0.100 4.571 ± 0.530 10.710 ± 1.240 0.13 ± 0.002 0.10 ± 0.001
0.020 ± 0.001 0.090 ± 0.003 0.170 ± 0.020 0.380 ± 0.040 4.220 ± 0.420 9.440 ± 1.560 0.20 ± 0.006 0.08 ± 0.002
0.015 ± 0.002 0.110 ± 0.020 0.22 ± 0.020 0.410 ± 0.030 3.730 ± 0.420 14.667 ± 1.520 0.33 ± 0.004 0.06 ± 0.001
0.022 ± 0.006 0.130 ± 0.030 0.090 ± 0.010 0.230 ± 0.020 1.769 ± 0.240 4.070 ± 0.370 0.12 ± 0.002 0.05 ± 0.002
0.004 ± 0.003 0.010 ± 0.002 0.020 ± 0.003 0.060 ± 0.005 6.000 ± 0.560 5.000 ± 0.540 0.090 ± 0.002 0.02 ± 001
STRS-SLNs STRS-SLNs/F-STRS STRS-SLNs/F-STRS F-STRS STRS-SLNs
There is statistically significant difference (p < 0.001) in %radioactivity of F-STRS and STRS-SLNs in blood, brain and kidney of mice at all the time points.
Table 5 Pharmacokinetic parameters in blood and brain after intranasal administration of 99m Tc labeled F-STRS and STRS-SLNs to mice (n = 4). Parameters
F-STRS
Cmax (%radioactivity/g) Tmax (h) AUC 0–∞ (%radioactivity/g) AUMC 0–∞ (%radioactivity/g) MRT (h) Vd (L) CL (L/h)
STR-SLNs
Blood
Brain
Blood
Brain
0.02 ± 0.01 4.00 ± 0.025 0.49 ± 0.010 5.65 ± 0.018 6.03 ± 0.56 0.066 ± 0.002 0.007 ± 0.001
0.13 ± 0.01 4.00 ± 0.02 1.91 ± 0.62 11.34 ± 0.012 5.99 ± 0.77 0.011 ± 0.001 0.002 ± 0.001
0.22 ± 0.02 2.00 ± 0.03 3.78 ± 0.60 58.30 ± 2.50 15.39 ± 1.10 0.009 ± 0.001 0.001 ± 0.001
0.41 ± 0.02 2.00 ± 0.01 6.83 ± 0.52 66.00 ± 3.50 9.66 ± 0.82 0.004 ± 0.00 0.0005 ± 0.0001
There is statistically significant difference (p < 0.001) in all the parameters of F-STRS and STRS-SLNs in blood and brain of mice.
6.10. Gamma scintigraphy In order to visualize comprehensive biodistribution following IN administration of 99m Tc-STRS-SLNs and F-STRS, we used a gamma scintigraphy camera. The gamma scintigraphic images of rabbits, 1 h post IN administration are shown in Fig. 7. 99m Tc-STRS-SLNs had a significant uptake in the brain tissue (estimated count of 489 ± 12 at the region of interest as compared to 123 ± 7 in case of the F-STRS). Results obtained are indicative of a capacity of STRSSLNs to effectively deliver drug to the brain. Furthermore, negligible amounts of radioactivity in the kidney at the end of 1 h, after administration of STRS-SLNS as compared to F-STRS are indicative of the slow and controlled renal excretory route of STRS-SLNs. Significant radioactivity was also observed in stomach of animals with the amount of radioactivity being substantially high in case of STRSSLNs, this could be due to the pouring of F-STRS or STRS-SLNs into
stomach via the nasopharyngeal duct. As it is reported that acidic pH (of stomach) leads to ionization of STRS (Berkman et al., 1947) so observation of lower radioactivity in case of F-STRS in stomach may indirectly indicate that STRS-SLNs protect STRS against degradation at acidic pH due to its encapsulation within SLNs, while F-STRS degrades resulting in the loss of radioactivity too. A similar protection to rifampicin against its acid induced degradation upon incorporation into SLNs has been reported by us (Singh et al., 2013). Furthermore, uptake of intact SLNs from the stomach and upper intestines and their transport into the brain has been reported by us earlier (Kakkar and Kaur, 2011; Kakkar et al., 2011, 2013b; Bhandari and Kaur, 2013). A similar phenomenon may be helping in the reuptake of these SLNs (being poured into the gut via nasopharyngeal pathway) thus resulting in their higher concentration in the brain. Gamma scintigraphs (Fig. 7) in New Zealand rabbits as shown by a four times higher radioactivity (489 versus 123) at the ROI (brain),
Fig. 7. Gamma scintigraphic images of New Zealand rabbit 1 h post IN administration of a) STRS-SLNs b) F-STRS.
232
M. Kumar et al. / International Journal of Pharmaceutics 461 (2014) 223–233
confirmed the results obtained with the biodistribution studies, highlighting the potential of STRS-SLNs for intranasal drug delivery. 7. Conclusions Drug agents having a poor intrinsic permeability and or stability issues upon oral administration can be redesigned using suitable carrier system and an alternative route of administration to achieve the desired pharmacodynamic effects. Presently, we intercept the limitations to the use of streptomycin, as an effective antitubercular agent using solid lipid nanoparticles as the proposed delivery system. Further, intranasal delivery is explored as an alternative to invasive parenteral route. The latter can improve patient compliance with therapy and the use of SLNs can result in achieving better or similar effect with the same or lower dose. Reducing the dose or plasma concentration of free drug (most of the drug in circulation will be in the encapsulated form acting as a depot from which free drug is released slowly and over a prolonged period) is of utmost importance in case of streptomycin, considering the severe and irreversible side effects reported with its use. These side effects are directly associated with the concentration of free drug in plasma (Berkman et al., 1947). Use of STRS-SLNs may also overcome the problem of limiting its use to not more than 2–3 months in the complete antitubercular therapeutic regimen of almost 9 months to one and half year for tubercular meningitis. It may be noted that a major concern with streptomycin is the induction of resistance, which is primarily due to its poor cytoplasmic permeability across the mycobacterial plasma membrane (Chambers, 2005). SLNs can overcome this barrier to STRS permeability, thus assigning it with an improved susceptibility of Mycobacterium and lowered incidence of producing resistant strains. Further, it is hypothesized that these nanoantibiotics (STRS-SLNs) are likely to enter into and act on the Mycobacterium by a variety of mechanisms, and it is impossible for the organism to induce mutations at so many sites (Huh and Kwon, 2011). This study may help to elevate the potential of STRS in antitubercular armamentarium due to its expected reduction in toxicity and improved permeation at lower doses. Nonetheless, more extensive mechanistic studies are warranted in this area, to confirm the potential of these nano delivery systems on pathogens. Declaration of interest The authors report no declarations of interest. Acknowledgments The contingent grant provided by DBT, New Delhi, India, and gift samples provided by Ranbaxy research laboratories, Gurgaon, India; Colorcon Asia Pacific Pvt. Ltd., Singapore and Panacea Biotec Lalru, Panjab, India are highly acknowledged. Use of TEM and XRD at the SAIF facility of Panjab University, Chandigarh, India, is duly acknowledged References Awasthi, S., Hallene, L.K., Fazio, V., Singhal, S.S., Luca, C., Awasthi, Y.C., Dine, G., Janigro, D., 2005. RLIP76, a non-ABC transporter, and drug resistance in epilepsy. BMC Neurosci. 6, 61. Banerjee, S.K., Jagannath, C., Hunter, R.L., Dasgupta, A., 2000. Bioavailability of tobramycin after oral delivery in FVB mice using CRL-1605 copolymer, an inhibitor of P-glycoprotein. Life Sci. 67, 2011–2016. Barakat, N.S., Omar, S.A., Ahmed, A.A., 2006. Carbamazepine uptake into rat brain following intra-olfactory transport. J. Pharm. Pharmacol. 58, 63–72. Berkman, S., Richard, H., Houseright, R., 1947. Studies on streptomycin I Factors influencing activity of streptomycin. J. Bacteriol. 53, 567–574. Bhandari, R., Kaur, I.P., 2013. Pharmacokinetics, tissue distribution and relative bioavailability of isoniazid-solid lipid nanoparticles. Int. J. Pharm. 441, 202–212.
Cavalli, R., Caputo, O., Carlotti, M.E., Trotta, M., Scarnecchia, C., Gasco, M.R., 1997. Sterilization and freeze drying of drug-free and drug-loaded solid lipid nanoparticles. Int. J. Pharm. 148, 47–54. Chambers, H.F., 2005. Antimicrobial agents. In: Brunton, L.L., Lazo, J.S., Parker, K.L. (Eds.), Goodman and Gillman’s the Pharmcological Basis of Therapeutics. McGrawHill Publication, pp. 1219–1234. Choi, A.O., Maysinger, D., 2013. Intranasal fluorescent nanocrystals for longitudinal in vivo evaluation of cerebral microlesions. Pharm. Nanotech. 1, 93–104. Costantino, H., Illum, L., Brandt, G., Johnson, P., Quay, S., 2007. Intranasal delivery: physicochemical and therapeutic aspects. Int. J. Pharm. 337, 1–24. Davis, S.S., Hardy, J.G., Newman, S.P., Wilding, I.R., 1992. Gamma scintigraphy in the evaluation of pharmaceutical dosage forms. Eur. J. Nucl. Med. Mol. Imaging 19, 971–986. Digenis, G.A., 1982. In radionuclide imaging in drug research. J. R. Soc. Med. 75, 683. Dorman, D.C., Brenneman, K.A., McElveen, A.M., Lynch, S.E., Roberts, K.C., Wong, B.A., 2002. Olfactory transport: a direct route of delivery of inhaled manganese manganese phosphate to the rat brain. J. Toxicol. Environ. Health A65, 1493–1511. Dragphia, R., Caillaud, C., Manicom, R., Pavirani, A., Kahn, A., Poenaru, L., 1995. Gene delivery into the central nervous system by nasal instillation in rats. Gene Ther. 2, 418–423. Garcia-Fuentes, M., Torres, D., Alonso, M.J., 2005. New surface-modified lipid nanoparticles as delivery vehicles for salmon calcitonin. Int. J. Pharm. 296, 122–132. Gasco, M.R., 1993. Method for producing solid lipid microspheres having a narrow size distribution. US Patent 5250236. Gasco, M.R., 1997. Solid lipid nanospheres from warm microemulsions. Pharm. Technol. Eur. 9, 52–58. Gohla, S.H., Dingler, A., 2001. Scaling up feasibility of the production of solid lipid nanoparticles (SLNTM ). Pharmazie 56, 61–63. Grassin-Delyle, S., Buenestado, A., Naline, E., Faisy, C., Blouquit-Laye, S., Couderc, L.-J., Le Guen, M., Fischler, M., Devillier, P., 2012. Intranasal drug delivery: an efficient and non-invasive route for systemic administration: focus on opioids. Pharmacol. Ther. 134, 366–379. Gupta, H., Aqil, M., Khar, R.K., Ali, A., Bhatnagar, A., Mittal, G., Jain, S., 2009. Development and characterization of 99mTc-timolol maleate for evaluating efficacy of in situ ocular drug delivery system. AAPS PharmSciTech 10, 540–546. Hanson, H.R., Frey, W.H., 2008. Intranasal delivery bypasses the blood-brain barrier to target therapeutic agents to the central nervous system and treat neurodegenerative disease. BMC Neurosci. 9, S5. Huh, A.J., Kwon, Y.J., 2011. Nanoantibiotics: a new paradigm for treating infectious diseases using nanomaterials in the antibiotics resistant era. J. Control. Release 156, 128–145. Illum, L., 2000. Transport of drugs from the nasal cavity to central nervous system. Eur. J. Pharm. Sci. 11, 1–18. Illum, L., 2003. Nasal drug delivery: problems, possibilities and solutions. J. Control. Release 87, 187–198. Kakkar, V., Kaur, I.P., 2011. Evaluating potential of curcumin loaded solid lipid nanoparticles in aluminium induced behavioural, biochemical and histopathological alterations in mice brain. Food Chem. Toxicol. 49, 2906–2913. Kakkar, V., Mishra, A., Chuttani, K., Chopra, K., Kaur, I.P., 2011. Delivery of sesamol loaded solid lipid nanoparticles to brain for menopause related emotional and cognitive central nervous system derangements. Rejuv. Res. 14, 597–604. Kakkar, V., Mishra, A.K., Chuttani, K., Kaur, I.P., 2013a. Proof of concept studies to confirm the delivery of curcumin loaded solid lipid nanoparticles (C-SLNs) to brain. Int. J. Pharm. 448, 354–359. Kakkar, V., Muppu, S.K., Chopra, K., Kaur, I.P., 2013b. Curcumin loaded solid lipid nanoparticles: an efficient formulation approach for cerebral ischemic reperfusion injury in rats. Eur. J. Pharm. Biopharm., http://dx.doi.org/10.1016/j.ejpb.2013.02.005. Kaur, I.P., Bhandari, R., Bhandari, S., Kakkar, V., 2008. Potential of solid lipid nanoparticles for brain targeting. J. Control. Release 127, 97–109. Kaur, I.P., Singh, M., Verma, M.K., 2012. Oral nano colloidal aqueous dispersion (NCD) of streptomycin sulphate In: 3093/DEL/2012, I.P.A. (Ed). India. Kaur, I.P., Singh, M., Verma, M.K., 2012b. Need to reformulate Streptomycin for unconventional routes of administration – an overview. In: Ray, A., Gupta, K. (Eds.), Translational Research in New Drug Development, New Delhi. , pp. 367–384. Kisel, M.A., Kulik, L.N., Tsybovsky, I.S., Vlasov, A.P., Vorob’yov, M.S., Kholodova, E.A., Zabarovskaya, Z.V., 2001. Liposomes with phosphatidylethanol as a carrier for oral delivery of insulin: studies in the rat. Int. J. Pharm. 216, 105–114. Kuo, Y.-C., Chung, C.-Y., 2011. Solid lipid nanoparticles comprising internal Compritol 888 ATO, tripalmitin and cacao butter for encapsulating and releasing stavudine, delavirdine and saquinavir. Colloids Surf. B 88, 682–690. Li, Q., Gao, M.L.X., 2008. A novel method for the determination of streptomycin using sodium nitroprusside as a chromogenic reagent by spectrophotometry. Anal. Lett. 41, 2595–2607. Manjunath, K., Reddy, J.S., Venkateswarlu, V., 2005. Solid lipid nanoparticles as drug delivery systems. Methods Find. Exp. Clin. Pharmacol. 27, 127–144. Müller, R.H., Runge, S., Ravelli, V., Mehnert, W., Thunemann, A.F., Souto, E.B., 2006. Oral bioavailability of cyclosporine: solid lipid nanoparticles (SLN) versus drug nanocrystals. Int. J. Pharm. 317, 82–89. Orecchioni, A.M., Duclairior, C., Depraetere, P., Osterstock, F., Nakache, E., 2003. Evaluation of gliadins nanoparticles as drug delivery systems a study of three different drugs. Int. J. Pharm. 253, 133–144.
M. Kumar et al. / International Journal of Pharmaceutics 461 (2014) 223–233 Pandey, R., Khuller, G.K., 2007. Nanoparticle-based oral drug delivery system for an injectable antibiotic –streptomycin,evaluation in a murine tuberculosis model. Chemotherapy 53, 437–441. Sanchez-Silos, R.M., Perez-Giraldo, C., Gomez-Garccia, A.C., 2001. Resistance to vancomycin, LY33328, ciprofloxacin and Trovofloxacin of community acquired and nosocomial strains of Enterococcus faecalis isolated in Badajoz (Spain) with and without high-level resistance to streptomycin and gentamycin. Chemotherapy 47, 415–420. Singh, H., Bhandari, R., Kaur, I.P., 2013. Encapsulation of Rifampicin in a solid lipid nanoparticulate system to limit its degradation and interaction with Isoniazid at acidic pH. Int. J. Pharm. 446, 106–111. Thorne, R.G., Hanson, L.R., Ross, T.M., Tung, D., Frey, W.H., 2008. Delivery of interferon beta to the monkey nervous system following intranasal administration. Neuroscience 152, 785e797. Uner, M., 2006. Preparation, characterization and physico-chemical properties of solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC): their benefits as colloidal drug carriers systems. Pharmazie 1, 375–386.
233
Verma, M.K., Singh, M., kakkar, V., Kaur, I.P., 2012. Applicability of a colorimetric method for evaluating streptomycin sulphate loaded solid lipid nanoparticles. Int. J. Pharm. Pharm. Sci. 4, 50–53. Vyas, T.K., Shahiwala, A., Marathe, S., Misra, A., 2005. Intranasal drug delivery for brain targeting. Curr. Drug Deliv. 2, 164–175. Wermling, D.P., Miller, J.P., Archer, S.M., Manaligod, J.M., Rudy, A.C., 2001. Bioavailability and pharmacokinetics of lorazepam after intranasal, intravenous and intramuscular administration. J. Clin. Pharmacol. 41, 1225–1231. WHO, 2010. Treatment of Tuberculosis: Guidelines for National Programmes. In Vol. WHO/CDS/TB/2003.31. World Health Organization, Geneva. Wilding, I.R., Coupe, A.J., Davis, S.S., 2001. The role of [gamma]-scintigraphy in oral drug delivery. Adv. Drug Deliv. Rev. 46, 103–124. Zensi, A., Begley, D., Pontikis, C., Legros, C., Mihoreanu, L., Wagner, S., Büchel, C., Briesen, H.V., Kreuter, J., 2009. Albumin nanoparticles targeted with Apo E enter the CNS by transcytosis and are delivered to neurones. J. Control. Release 137, 78–86.
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
Intranasal delivery of streptomycin sulfate (STRS) loaded solid lipid nanoparticles to brain and blood Manoj Kumar a , Vandita Kakkar a , Anil Kumar Mishra b , Krishna Chuttani b , Indu Pal Kaur a,∗ a b
University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh 160014, India Institute of Nuclear Medicine and Allied Science (INMAS), Timarpur, New Delhi, India
a r t i c l e
i n f o
Article history: Received 8 August 2013 Received in revised form 8 November 2013 Accepted 18 November 2013 Available online 25 November 2013 Keywords: Streptomycin Intranasal Solid lipid nanoparticles Biodistribution Blood
a b s t r a c t Factors like unreliable and poor oral absorption, including an active Pgp-efflux point towards a compromised oral bioavailability (BA) of streptomycin sulfate (STRS). Latter instigates its parenteral use (i.m.) only. Furthermore, its chronic use leads to serious side effects like nephrotoxicity and ototoxicity. In the present study, we propose to develop streptomycin sulfate (STRS) loaded solid lipid nanoparticles (STRS-SLNs) for non-invasive intranasal (IN) delivery. STRS-SLNs were prepared using patented nanocolloidal aqueous dispersion technique (Indian Patent application 3093/DEL/2012). Small particle size (140.1 ± 7.0 nm) and significant entrapment efficiency (54.83 ± 2.1%) was achieved. Biodistribution studies using 99m Tc showed a 3.15 and 11.0 times higher concentrations in the brain and blood of mice, respectively, on IN administration of STRS-SLNs in comparison to free (F)-STRS. Lower concentrations (3.3 times) in kidneys implicate lower nephrotoxicity. Similarly a 12 and 4 times lower levels of drug in liver and spleen, respectively upon administration of STRS-SLNs as compared to F-STRS also indicate its lesser accumulation in these reticuloendothelial system organs. Lipophillic enclosure imparted to STRS, coupled with small particle size, and its purported ability to inhibit Pgp-efflux due to the presence of tween 80, is considered to be responsible for a better BA shown by STRS upon incorporation into SLNs. This is predicted to result in an effective treatment of all types of tuberculosis including cerebral tuberculosis as indicated by high relative distribution to brain in comparison to free-STRS. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Streptomycin (STR), an aminoglycoside, is a bactericidal antibiotic active against both gram-negative and gram-positive organisms, and Mycobacterium. It acts primarily by inhibition of protein synthesis leading to codon misreading and ultimately death of microbial cells (Chambers, 2005). Streptomycin sulfate (STRS) is indicated as a first line (though parenteral) antitubercular drug (ATD) and is especially suggested for (i) patients with hepatitis and other liver disorders induced by other hepatotoxic oral ATDs, viz. rifampicin, isoniazid and pyrazinamide. A non-hepatotoxic regimen consisting of streptomycin, ethambutol and a fluoroquinolone is recommended in such situations, for 18–24 months with or without 1 or 2 hepatotoxic ATDs; (ii) tuberculous meningitis, where ethambutol is suggested to be replaced by streptomycin; and, (iii) patients returning after default or relapse and now receiving the
∗ Corresponding author at: Department of Pharmaceutics, University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh-160014, India. Tel.: +91 172 2534191; fax: +91 172 2543101. E-mail address: [email protected] (I.P. Kaur). 0378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2013.11.038
8-month retreatment regimen, with an initial 2 months of intensive treatment with all first line drugs including streptomycin (WHO, 2010). Streptomycin suffers from serious risk of nephrotoxic reactions in patients with impaired kidney function; irreversible ototoxicity is also reported at high doses. Intranasal (IN) delivery is considered a promising alternative to intravenous route for a rapid delivery of therapeutic agents to the blood and brain for treating CNS disorders (Costantino et al., 2007; Hanson and Frey, 2008; Grassin-Delyle et al., 2012). It has been demonstrated that part of the therapeutic administered via the intranasal route may be delivered directly to the CNS along both the olfactory and trigeminal nerve (Thorne et al., 2008). IN route provides advantages including a large surface area for absorption, rapid achievement of target drug levels, avoidance of first pass metabolism and improvement of drug bioavailability (BA). Furthermore, this delivery route, in comparison to the parenteral route shows maximal patient comfort and compliance (Dragphia et al., 1995; Illum, 2000, 2003; Wermling et al., 2001; Dorman et al., 2002; Vyas et al., 2005). However, the rapid removal of formulation by mucociliary clearance often limits the BA of drugs delivered intranasally. Therefore, retaining drug longer in the nasal cavity using options like mucoadhesives or nanoparticulate
224
M. Kumar et al. / International Journal of Pharmaceutics 461 (2014) 223–233
systems can help maximize the utility of IN delivery. In addition to the prolonged drug residence time, for poorly absorbed drugs such as streptomycin, nanoparticulate systems can help to enhance the permeability across the epithelial membrane. Latter will be essential for achieving improved systemic delivery (Garcia-Fuentes et al., 2005; Müller et al., 2006). Solid lipid nanoparticles (SLNs) are drug carriers with a particle size 50% drug within the SLN/NCD system, considering the highly hydrophilic nature of STRS as indicated by its very high negative log p of −6.7. Though hydrophilic drugs are proposed to be incorporated into SLNs but achieving significant payload and entrapment efficiency into the lipophilic carrier system is an issue (Kisel et al., 2001; Bhandari and Kaur, 2013). Novelty of the developed NCD lies both in the method of preparation and in the fact that more concentrated nanoparticles/volume are achieved by the present method, in comparison to the corresponding SLN dispersions prepared using a generally employed microemulsion method. Latter involves a 1:10–1:100 dilution of the microemulsion with cold water (Gasco, 1993, 1997; Gohla and Dingler, 2001). Such dilute dispersions are recommended to be lyophilized and reconstituted before use (Gasco, 1993, 1997). Latter will invariably result in aggregates and hence some microsize particles rather than uniformly distributed nanostructures. Another process recommended for concentrating the dilute nanodispersions is diafiltration. Personally speaking, we have a strong reservation to the later method as it is in contrast to the very definition of SLNs which states that they constitute ‘lipid nanoparticles dispersed in an aqueous surfactant solution’. Upon diafiltration the surfactant present in the aqueous dispersion is removed and the remaining nanocolloidal dispersion will again tend to aggregate and will be destabilized. Presence of surfactant molecules in the aqueous medium is very critical to the stability of the nanosystem and are specific to that system. Hence, it is highlighted here that the SLN/NCD system presently reported for the encapsulation of STRS is unique in the sense that inspite of a high solid content (8.8% which is 10 to 100 times higher than normally reported system) the developed system was very stable showing no significant change in size after storage of up to 2 years under refrigeration.
Fig. 1. TEM micrograph of STRS-SLNs.
Latter is attributable to maintaining the innate nature in which the system was developed. Furthermore the amount of drug which can be loaded/volume in the present system also tends to be high (due to lower dilution) and it is thus practical to administer clinically the otherwise high dose of such agents. 6.1. Particle size analysis Average particle size of STRS-SLNs was 140.1 ± 7.0 nm with a polydispersity index (P.I.) of 0.281, when measured using photon correlation spectroscopy (Malvern, Zetasizer UK). A small PI indicates uniformly produced nanodispersion; no micronized particles were observed in the entire population. The small particle size achieved with SLNs and encapsulation of STRS within lipidic core is expected to facilitate transport of drug across the nasal mucosal barriers both into the cerebral tissues and systemic circulation (Choi and Maysinger, 2013). 6.2. TEM TEM analysis revealed spherical particles in size range of the order 90–120 nm. The size of the nanoparticles observed under TEM (Fig. 1) was smaller than the results obtained using Mastersizer® . This may however be attributed to the fact, that Mastersizer® is based on the principle of dynamic light scattering, which at times may not detect small particles, due to the higher brownian movement of these particles. TEM on the other hand, involves observation of a fairly small number of representative particles (in 2–3 fields) of a dilute dispersion such that the particle size observed under TEM do not match with results obtained by the PCS technology which gives population statistics. 6.3. Total drug content (TDC) and entrapment efficiency (EE) TDC of STRS-SLNs was estimated to be 88.23 ± 1.2% (n = 6) and the EE of STRS-SLNs was 54.83 ± 2.1% (n = 6). Entrapment values of >50% for STRS which is a highly hydrophilic molecule (log p = −6.7) have never been reported earlier in the available literature. Pandey and Khuller (2007) have worked extensively on several antitubercular drugs including streptomycin. They report polymeric nanoparticles of STRS with an EE of 32%; particle size of the formulation prepared by them was 153 nm. Compritol® 888
M. Kumar et al. / International Journal of Pharmaceutics 461 (2014) 223–233
227
Fig. 2. DSC of a) STRS b) Compritol® 888 ATO c) STRS-SLNs.
ATO was presently chosen as the lipid component because it tends to result in dispersions with small particle size and also shows good distribution into brain (Cavalli et al., 1997; Kaur et al., 2008). Furthermore, the surfactant to co-surfactant, ratio was kept suitably high to achieve not only a small size but also a significant EE with a better brain distribution (Kakkar and Kaur, 2011; Kuo and Chung, 2011; Bhandari and Kaur, 2013; Kakkar et al., 2013b; Singh et al., 2013). 6.4. DSC DSC is a thermoanalytical technique in which the difference in the amount of heat required to maintain the sample and reference at the same temperature is measured as a function of temperature and time. The breakdown or fusion of the crystal lattice by heating or cooling the sample yields information about the internal polymorphism, crystal order, or glass transition processes (Uner, 2006). It uses the fact that different lipid modifications possess different melting points and enthalpies. The basic principle underlying this technique is that when the sample undergoes a physical
transformation (such as melting, desolvation), some amount of heat is required to flow to it depending on whether the process is exothermic or endothermic to maintain both the reference and sample at the same temperature. DSC measures this heat flow into or from the sample when it is heated or cooled and thus provides qualitative and quantitative information about physicochemical changes (i.e. endothermic or exothermic nature of the process or changes in heat capacity). In case of pure streptomycin sulfate, melting endotherm appeared at 151.81 ◦ C corresponding to its melting point and was associated with 252.3 J/g of enthalpy, while Compritol® 888 ATO showed a sharp peak at 74.72 ◦ C and an enthalpy of 122.6 J/g. STRSSLNs showed an endothermic shift to 98.77 ◦ C and a significantly lowered heat flow of 66.79 J/g (Fig. 2) indicating incorporation of drug into the lipid matrix. A significant lowering of enthalpy for STRS-SLNs versus pure lipid indicates a reduction in particle size and the change in polymorphic state of the lipid from its crystalline  form to the amorphous (␣,  ) form (Müller et al., 2006) with more imperfections in the crystal lattice. Latter results in comfortable incorporation of more amount of drug within the lipid molecules.
228
M. Kumar et al. / International Journal of Pharmaceutics 461 (2014) 223–233 Counts
Table 1 Peaks of FTIR spectra for STRS, Compritol® 888 ATO and STRS-SLNs.
400
200
0
10
Counts
20
30
40
Position [°2Theta] (Copper (Cu))
a) 6000
4000
2000
0
10
20
30
40
Position [°2Theta] (Copper (Cu))
b)
Sr no
STRS (cm−1 )
Compritol® 888 ATO (cm−1 )
STRS-SLNs (cm−1 )
1. 2. 3. 4. 5. 6 7. 8. 9. 10. 11.
3364.7 2933.5 1671.5 1460.5 1124.9 1052.9 862.6 618.5
3348.9 2919.5 1739.9 1624.9 1468.4 1382.1 1174.8 11097 1054.6 722.1 549.7
3432.3 2921.1 1736.8 1651.3 1463.2 1352.8 1251.3 1105.8 9506 838.7 538.5
3364.7 cm−1 (N–H stretching) in STRS spectra, 3448.9 cm−1 (due to –OH– stretch of –COOH group) in Compritol® 888 ATO and a similar stretch is observed in STRS-SLNs at 3432.3 cm−1 . It may however be noted that the peak due to –OH stretch is reported to be broad (as also observed by us, Fig. 4b), while the peak observed for prepared SLNs at 3432.3 cm−1 tends to be sharper.
Counts
6.7. In vitro release
1000
500
0
10
20
30
40
Position [°2Theta] (Copper (Cu))
c) Counts 600
400 200
0
10
d)
20
30
40
Position [°2Theta] (Copper (Cu))
Fig. 3. P-XRD of a) STRS b) Compritol® 888 ATO c) blank SLNs d) STRS-SLNs.
The drug release from STRS-SLNs at 37 ± 0.5 ◦ C is shown in Fig. 5. A 100% release of STRS from STRS-SLNs was achieved at the end of 24 h, while in case of the F-STRS complete release was observed within 2 h. The in vitro drug release from STRS-SLNs showed a phasic behavior. At initial time points of up to 1 h a superclass II release was observed. This may be ascribed to either a fast release of the free unentrapped drug (%EE of STRS-SLNs was 54.85% and the remaining 45.17% of free-STRS is also present in the SLN dispersion) across the dialysis membrane or burst release and/or erosion of the drug from the outer phospholipid/surfactant layer of SLNs as is expected for a hydrophilic drug (Bhandari and Kaur, 2013). Subsequent release (post 1 h), was non fickian i.e., a combination of diffusion and erosion. STRS is a highly water soluble drug and hence its adsorption on the surface of the lipidic nanoparticles and its consequent release into the dialysis bag coupled with the passage of very small SLNs across the dialysis bag may be responsible for the second fast release phase, starting from 1 h onwards till 8 h, where there is a hump (a further increase) up to 12 h. Of the various release models, Higuchi release model was best fitted for the developed solid lipid nanoparticles (Table 2) at all the time points.
6.5. Powder X-ray diffraction (PXRD)
6.8. Stability study
PXRD patterns of STRS, Compritol® 888 ATO, lyophilized blank SLNs (B-SLNs) and lyophilized STRS-SLNs are shown in Fig. 3. PXRD pattern of STRS shows an amorphous nature while the PXRD of Compritol® 888 ATO showed sharp peaks at 2 scattered angles 21.16, 23.37, 23.52 and 35.76, establishing its crystalline nature. However, absence of these characteristic peaks in lyophilized BSLNs and STRS-SLNs sample, confirm the loss of crystallinity of the lipid and its shift towards the amorphous state. Latter indicates probable incorporation of STRS into the lipid matrix of the SLNs in the final formulation.
After 2 years of storage at 5 ± 3 ◦ C, the STRS-SLN dispersion was found to be stable, with no significant increase in size and depicting 100% assay and entrapment efficiency (p < 0.001) with respect to initial values at the end of 2 years (Table 3). Part of this stability (as discussed earlier) may be attributed to the maintenance of SLNs in their native state in which they were produced comprising suitable concentrations of surfactant in the aqueous phase in which they are dispersed. Another reason may be attributed to the composition of SLNs including an appropriate surfactant: co-surfactant ratio and the method of preparation which results in a concentrated nanoparticle dispersion with high solid content.
6.6. FTIR spectroscopy The FTIR spectra for F-STRS, Compritol® 888 ATO and the developed STRS-SLNs are shown in Fig. 4. Closer inspection of various peaks (Table 1) obtained in these spectra indicate two new peaks at 1651.3 cm−1 (N–H bending) and at 1251.3 cm−1 (C–N stretching) in the STRS-SLNs spectra. These new peaks indicate probable incorporation of STRS in SLNs. Further to this a peak is observed at
6.9. In vivo biodistribution studies Biodistribution studies of STRS-SLNs and F-STRS in the brain and blood of mice following IN administration, were conducted using technetium labeled (99m Tc-labeled) free drug solution or its developed SLNs at different times post administration (Table 4). The
M. Kumar et al. / International Journal of Pharmaceutics 461 (2014) 223–233
229
Fig. 4. FTIR spectrum of a) STRS b) Compritol® 888 ATO c) STRS-SLNs.
results showed achievement of 3.15 and 11.0 times, higher radioactivity in brain and blood respectively when STRS-SLNs (Table 4) were administered IN, in contrast to F-STRS, thus confirming the efficacy of the developed system. There was 7.7 times and 10.31
times enhancement in AUC 0 − ∞ and AUMC 0 − ∞ for STRS-SLNs as compared to F-STRS in blood, and 3.5 times and 5.8 times in brain respectively. Further 2.55 times and 1.61 times enhanced mean residence time was observed for STRS-SLNs as compared to F-STRS
230
M. Kumar et al. / International Journal of Pharmaceutics 461 (2014) 223–233
Table 2 Invitro release kinetics. R2 in different release models
Time points (h)
Value of n
Mechanism of drug release
Korsmeyer peppas
Higuchi
First order
Zero order
0.25 0.5 1 2 4 8 12 24
n = 0.93
Super class II, erosion
0.9374
0.9617
0.8163
0.9187
n = 0.58
Non fickian diffusion and erosion controlled rate release
0.9994
0.9995
0.9736
0.9986
0.8591
0.8618
0.7568
0.8162
Table 3 Stability study parameters during storage of STRS-SLNs at 5 ± 3 ◦ C (n = 6). Time points
Av. particle size (nm)
Total drug content* (%)
Entrapment efficiency* (%)
0 times 6 months 12 months 24 months
141.0 ± 5.0 105.0 ± 12.0 125.0 ± 8.0* 144.1 ± 6.0*
89.0 ± 2.5 88.6 ± 1.2* 88.2 ± 1.6* 89.92 ± 2.4*
54.83 ± 2.1* 53.14 ± 1.5* 53.87 ± 1.2* 52.07 ± 1.8*
*
*
* Statistically insignificant difference (p < 0.001) in average particle size at 0, 12 and 24 months of stability samples. Further, no statistically significant difference (p < 0.001) in total drug content and entrapment efficiency was observed at any time point.
Fig. 5. In vitro drug release of F-STRS and STR-SLNs in phosphate buffer (pH 7.4). ANOVA indicates that values at one time point are significantly different (p < 0.001) from the preceeding and succeeding time points except where marked suitably (*).
in blood and brain, respectively. A faster tmax (2 h) obtained with SLNs in comparison to free drug (4 h) in the brain of the mice are indicative of the rapid transport of STRS-SLNs to the brain post IN administration (Table 5). STRS-SLNs showed 3.3 times lower concentrations of drug in kidney as compared to the free drug (Table 4) post intranasal administration. Similarly 12.0 and 4.0
times lesser drug concentrations were observed in liver and spleen tissues, respectively (Fig. 6). This may also indirectly indicate low incidence of nanoparticulate related toxicity, higher systemic availability and prolonged residence time (due to reduced metabolic conversion). Intranasal administration of STRS-SLNs is purported to bypass the RES organs, as a result of which their systemic availability and therefore their concentration across the BBB is expected to be higher in comparison to the poorly available free-STRS. Former was supported by the results obtained in the biodistribution studies in liver and spleen (Fig. 6). Furthermore improved brain bioavailability may be assigned to the use of tween 80 in the preparation of SLNs which is reported to improve brain delivery of nanoparticles by (i) solublization of endothelial cell membrane lipids and membrane fluidization, (ii) through the temporary opening of inulin spaces (Zensi et al., 2009), (iii) endocytosis of nanoparticles, and (iv) inhibition of efflux system, especially P-gp present on the intranasal membrane and also the BBB (Wermling et al., 2001; Awasthi et al., 2005). Furthermore, the lipidic nature of SLNs may also help in their preferred passage across the lipoidal biological membranes while the phospholipid coat may have an added advantage in assisting in their movement via paracellular pathways (Barakat et al., 2006).
Fig. 6. % Radioactivity/gram tissue in liver and spleen post administration of free drug and STRS-SLNs.
M. Kumar et al. / International Journal of Pharmaceutics 461 (2014) 223–233
231
Table 4 Biodistribution of 99m Tc-F-STRS and 99m Tc-STRS-SLNs in blood, brain and kidney at different time intervals after intranasal (IN) administration in Balb/c mice. Concentration of STRS (% radioactivity/g at different times) and place this title above the various time points. Formulation and route of administration Intransal (IN)
Organ/tissue
0.25 h
0.5 h
1h
2h
4h
24 h
Free drug (F-STRS)
Blood Brain Blood Brain Brain Blood Kidney Kidney
0.017 ± 0.002 0.070 ± 0.006 0.120 ± 0.003 0.080 ± 0.003 1.142 ± 0.160 7.059 ± 0.530 0.20 ± 0.003 0.09 ± 0.002
0.014 ± 0.005 0.085 ± 0.003 0.150 ± 0.060 0.320 ± 0.100 4.571 ± 0.530 10.710 ± 1.240 0.13 ± 0.002 0.10 ± 0.001
0.020 ± 0.001 0.090 ± 0.003 0.170 ± 0.020 0.380 ± 0.040 4.220 ± 0.420 9.440 ± 1.560 0.20 ± 0.006 0.08 ± 0.002
0.015 ± 0.002 0.110 ± 0.020 0.22 ± 0.020 0.410 ± 0.030 3.730 ± 0.420 14.667 ± 1.520 0.33 ± 0.004 0.06 ± 0.001
0.022 ± 0.006 0.130 ± 0.030 0.090 ± 0.010 0.230 ± 0.020 1.769 ± 0.240 4.070 ± 0.370 0.12 ± 0.002 0.05 ± 0.002
0.004 ± 0.003 0.010 ± 0.002 0.020 ± 0.003 0.060 ± 0.005 6.000 ± 0.560 5.000 ± 0.540 0.090 ± 0.002 0.02 ± 001
STRS-SLNs STRS-SLNs/F-STRS STRS-SLNs/F-STRS F-STRS STRS-SLNs
There is statistically significant difference (p < 0.001) in %radioactivity of F-STRS and STRS-SLNs in blood, brain and kidney of mice at all the time points.
Table 5 Pharmacokinetic parameters in blood and brain after intranasal administration of 99m Tc labeled F-STRS and STRS-SLNs to mice (n = 4). Parameters
F-STRS
Cmax (%radioactivity/g) Tmax (h) AUC 0–∞ (%radioactivity/g) AUMC 0–∞ (%radioactivity/g) MRT (h) Vd (L) CL (L/h)
STR-SLNs
Blood
Brain
Blood
Brain
0.02 ± 0.01 4.00 ± 0.025 0.49 ± 0.010 5.65 ± 0.018 6.03 ± 0.56 0.066 ± 0.002 0.007 ± 0.001
0.13 ± 0.01 4.00 ± 0.02 1.91 ± 0.62 11.34 ± 0.012 5.99 ± 0.77 0.011 ± 0.001 0.002 ± 0.001
0.22 ± 0.02 2.00 ± 0.03 3.78 ± 0.60 58.30 ± 2.50 15.39 ± 1.10 0.009 ± 0.001 0.001 ± 0.001
0.41 ± 0.02 2.00 ± 0.01 6.83 ± 0.52 66.00 ± 3.50 9.66 ± 0.82 0.004 ± 0.00 0.0005 ± 0.0001
There is statistically significant difference (p < 0.001) in all the parameters of F-STRS and STRS-SLNs in blood and brain of mice.
6.10. Gamma scintigraphy In order to visualize comprehensive biodistribution following IN administration of 99m Tc-STRS-SLNs and F-STRS, we used a gamma scintigraphy camera. The gamma scintigraphic images of rabbits, 1 h post IN administration are shown in Fig. 7. 99m Tc-STRS-SLNs had a significant uptake in the brain tissue (estimated count of 489 ± 12 at the region of interest as compared to 123 ± 7 in case of the F-STRS). Results obtained are indicative of a capacity of STRSSLNs to effectively deliver drug to the brain. Furthermore, negligible amounts of radioactivity in the kidney at the end of 1 h, after administration of STRS-SLNS as compared to F-STRS are indicative of the slow and controlled renal excretory route of STRS-SLNs. Significant radioactivity was also observed in stomach of animals with the amount of radioactivity being substantially high in case of STRSSLNs, this could be due to the pouring of F-STRS or STRS-SLNs into
stomach via the nasopharyngeal duct. As it is reported that acidic pH (of stomach) leads to ionization of STRS (Berkman et al., 1947) so observation of lower radioactivity in case of F-STRS in stomach may indirectly indicate that STRS-SLNs protect STRS against degradation at acidic pH due to its encapsulation within SLNs, while F-STRS degrades resulting in the loss of radioactivity too. A similar protection to rifampicin against its acid induced degradation upon incorporation into SLNs has been reported by us (Singh et al., 2013). Furthermore, uptake of intact SLNs from the stomach and upper intestines and their transport into the brain has been reported by us earlier (Kakkar and Kaur, 2011; Kakkar et al., 2011, 2013b; Bhandari and Kaur, 2013). A similar phenomenon may be helping in the reuptake of these SLNs (being poured into the gut via nasopharyngeal pathway) thus resulting in their higher concentration in the brain. Gamma scintigraphs (Fig. 7) in New Zealand rabbits as shown by a four times higher radioactivity (489 versus 123) at the ROI (brain),
Fig. 7. Gamma scintigraphic images of New Zealand rabbit 1 h post IN administration of a) STRS-SLNs b) F-STRS.
232
M. Kumar et al. / International Journal of Pharmaceutics 461 (2014) 223–233
confirmed the results obtained with the biodistribution studies, highlighting the potential of STRS-SLNs for intranasal drug delivery. 7. Conclusions Drug agents having a poor intrinsic permeability and or stability issues upon oral administration can be redesigned using suitable carrier system and an alternative route of administration to achieve the desired pharmacodynamic effects. Presently, we intercept the limitations to the use of streptomycin, as an effective antitubercular agent using solid lipid nanoparticles as the proposed delivery system. Further, intranasal delivery is explored as an alternative to invasive parenteral route. The latter can improve patient compliance with therapy and the use of SLNs can result in achieving better or similar effect with the same or lower dose. Reducing the dose or plasma concentration of free drug (most of the drug in circulation will be in the encapsulated form acting as a depot from which free drug is released slowly and over a prolonged period) is of utmost importance in case of streptomycin, considering the severe and irreversible side effects reported with its use. These side effects are directly associated with the concentration of free drug in plasma (Berkman et al., 1947). Use of STRS-SLNs may also overcome the problem of limiting its use to not more than 2–3 months in the complete antitubercular therapeutic regimen of almost 9 months to one and half year for tubercular meningitis. It may be noted that a major concern with streptomycin is the induction of resistance, which is primarily due to its poor cytoplasmic permeability across the mycobacterial plasma membrane (Chambers, 2005). SLNs can overcome this barrier to STRS permeability, thus assigning it with an improved susceptibility of Mycobacterium and lowered incidence of producing resistant strains. Further, it is hypothesized that these nanoantibiotics (STRS-SLNs) are likely to enter into and act on the Mycobacterium by a variety of mechanisms, and it is impossible for the organism to induce mutations at so many sites (Huh and Kwon, 2011). This study may help to elevate the potential of STRS in antitubercular armamentarium due to its expected reduction in toxicity and improved permeation at lower doses. Nonetheless, more extensive mechanistic studies are warranted in this area, to confirm the potential of these nano delivery systems on pathogens. Declaration of interest The authors report no declarations of interest. Acknowledgments The contingent grant provided by DBT, New Delhi, India, and gift samples provided by Ranbaxy research laboratories, Gurgaon, India; Colorcon Asia Pacific Pvt. Ltd., Singapore and Panacea Biotec Lalru, Panjab, India are highly acknowledged. Use of TEM and XRD at the SAIF facility of Panjab University, Chandigarh, India, is duly acknowledged References Awasthi, S., Hallene, L.K., Fazio, V., Singhal, S.S., Luca, C., Awasthi, Y.C., Dine, G., Janigro, D., 2005. RLIP76, a non-ABC transporter, and drug resistance in epilepsy. BMC Neurosci. 6, 61. Banerjee, S.K., Jagannath, C., Hunter, R.L., Dasgupta, A., 2000. Bioavailability of tobramycin after oral delivery in FVB mice using CRL-1605 copolymer, an inhibitor of P-glycoprotein. Life Sci. 67, 2011–2016. Barakat, N.S., Omar, S.A., Ahmed, A.A., 2006. Carbamazepine uptake into rat brain following intra-olfactory transport. J. Pharm. Pharmacol. 58, 63–72. Berkman, S., Richard, H., Houseright, R., 1947. Studies on streptomycin I Factors influencing activity of streptomycin. J. Bacteriol. 53, 567–574. Bhandari, R., Kaur, I.P., 2013. Pharmacokinetics, tissue distribution and relative bioavailability of isoniazid-solid lipid nanoparticles. Int. J. Pharm. 441, 202–212.
Cavalli, R., Caputo, O., Carlotti, M.E., Trotta, M., Scarnecchia, C., Gasco, M.R., 1997. Sterilization and freeze drying of drug-free and drug-loaded solid lipid nanoparticles. Int. J. Pharm. 148, 47–54. Chambers, H.F., 2005. Antimicrobial agents. In: Brunton, L.L., Lazo, J.S., Parker, K.L. (Eds.), Goodman and Gillman’s the Pharmcological Basis of Therapeutics. McGrawHill Publication, pp. 1219–1234. Choi, A.O., Maysinger, D., 2013. Intranasal fluorescent nanocrystals for longitudinal in vivo evaluation of cerebral microlesions. Pharm. Nanotech. 1, 93–104. Costantino, H., Illum, L., Brandt, G., Johnson, P., Quay, S., 2007. Intranasal delivery: physicochemical and therapeutic aspects. Int. J. Pharm. 337, 1–24. Davis, S.S., Hardy, J.G., Newman, S.P., Wilding, I.R., 1992. Gamma scintigraphy in the evaluation of pharmaceutical dosage forms. Eur. J. Nucl. Med. Mol. Imaging 19, 971–986. Digenis, G.A., 1982. In radionuclide imaging in drug research. J. R. Soc. Med. 75, 683. Dorman, D.C., Brenneman, K.A., McElveen, A.M., Lynch, S.E., Roberts, K.C., Wong, B.A., 2002. Olfactory transport: a direct route of delivery of inhaled manganese manganese phosphate to the rat brain. J. Toxicol. Environ. Health A65, 1493–1511. Dragphia, R., Caillaud, C., Manicom, R., Pavirani, A., Kahn, A., Poenaru, L., 1995. Gene delivery into the central nervous system by nasal instillation in rats. Gene Ther. 2, 418–423. Garcia-Fuentes, M., Torres, D., Alonso, M.J., 2005. New surface-modified lipid nanoparticles as delivery vehicles for salmon calcitonin. Int. J. Pharm. 296, 122–132. Gasco, M.R., 1993. Method for producing solid lipid microspheres having a narrow size distribution. US Patent 5250236. Gasco, M.R., 1997. Solid lipid nanospheres from warm microemulsions. Pharm. Technol. Eur. 9, 52–58. Gohla, S.H., Dingler, A., 2001. Scaling up feasibility of the production of solid lipid nanoparticles (SLNTM ). Pharmazie 56, 61–63. Grassin-Delyle, S., Buenestado, A., Naline, E., Faisy, C., Blouquit-Laye, S., Couderc, L.-J., Le Guen, M., Fischler, M., Devillier, P., 2012. Intranasal drug delivery: an efficient and non-invasive route for systemic administration: focus on opioids. Pharmacol. Ther. 134, 366–379. Gupta, H., Aqil, M., Khar, R.K., Ali, A., Bhatnagar, A., Mittal, G., Jain, S., 2009. Development and characterization of 99mTc-timolol maleate for evaluating efficacy of in situ ocular drug delivery system. AAPS PharmSciTech 10, 540–546. Hanson, H.R., Frey, W.H., 2008. Intranasal delivery bypasses the blood-brain barrier to target therapeutic agents to the central nervous system and treat neurodegenerative disease. BMC Neurosci. 9, S5. Huh, A.J., Kwon, Y.J., 2011. Nanoantibiotics: a new paradigm for treating infectious diseases using nanomaterials in the antibiotics resistant era. J. Control. Release 156, 128–145. Illum, L., 2000. Transport of drugs from the nasal cavity to central nervous system. Eur. J. Pharm. Sci. 11, 1–18. Illum, L., 2003. Nasal drug delivery: problems, possibilities and solutions. J. Control. Release 87, 187–198. Kakkar, V., Kaur, I.P., 2011. Evaluating potential of curcumin loaded solid lipid nanoparticles in aluminium induced behavioural, biochemical and histopathological alterations in mice brain. Food Chem. Toxicol. 49, 2906–2913. Kakkar, V., Mishra, A., Chuttani, K., Chopra, K., Kaur, I.P., 2011. Delivery of sesamol loaded solid lipid nanoparticles to brain for menopause related emotional and cognitive central nervous system derangements. Rejuv. Res. 14, 597–604. Kakkar, V., Mishra, A.K., Chuttani, K., Kaur, I.P., 2013a. Proof of concept studies to confirm the delivery of curcumin loaded solid lipid nanoparticles (C-SLNs) to brain. Int. J. Pharm. 448, 354–359. Kakkar, V., Muppu, S.K., Chopra, K., Kaur, I.P., 2013b. Curcumin loaded solid lipid nanoparticles: an efficient formulation approach for cerebral ischemic reperfusion injury in rats. Eur. J. Pharm. Biopharm., http://dx.doi.org/10.1016/j.ejpb.2013.02.005. Kaur, I.P., Bhandari, R., Bhandari, S., Kakkar, V., 2008. Potential of solid lipid nanoparticles for brain targeting. J. Control. Release 127, 97–109. Kaur, I.P., Singh, M., Verma, M.K., 2012. Oral nano colloidal aqueous dispersion (NCD) of streptomycin sulphate In: 3093/DEL/2012, I.P.A. (Ed). India. Kaur, I.P., Singh, M., Verma, M.K., 2012b. Need to reformulate Streptomycin for unconventional routes of administration – an overview. In: Ray, A., Gupta, K. (Eds.), Translational Research in New Drug Development, New Delhi. , pp. 367–384. Kisel, M.A., Kulik, L.N., Tsybovsky, I.S., Vlasov, A.P., Vorob’yov, M.S., Kholodova, E.A., Zabarovskaya, Z.V., 2001. Liposomes with phosphatidylethanol as a carrier for oral delivery of insulin: studies in the rat. Int. J. Pharm. 216, 105–114. Kuo, Y.-C., Chung, C.-Y., 2011. Solid lipid nanoparticles comprising internal Compritol 888 ATO, tripalmitin and cacao butter for encapsulating and releasing stavudine, delavirdine and saquinavir. Colloids Surf. B 88, 682–690. Li, Q., Gao, M.L.X., 2008. A novel method for the determination of streptomycin using sodium nitroprusside as a chromogenic reagent by spectrophotometry. Anal. Lett. 41, 2595–2607. Manjunath, K., Reddy, J.S., Venkateswarlu, V., 2005. Solid lipid nanoparticles as drug delivery systems. Methods Find. Exp. Clin. Pharmacol. 27, 127–144. Müller, R.H., Runge, S., Ravelli, V., Mehnert, W., Thunemann, A.F., Souto, E.B., 2006. Oral bioavailability of cyclosporine: solid lipid nanoparticles (SLN) versus drug nanocrystals. Int. J. Pharm. 317, 82–89. Orecchioni, A.M., Duclairior, C., Depraetere, P., Osterstock, F., Nakache, E., 2003. Evaluation of gliadins nanoparticles as drug delivery systems a study of three different drugs. Int. J. Pharm. 253, 133–144.
M. Kumar et al. / International Journal of Pharmaceutics 461 (2014) 223–233 Pandey, R., Khuller, G.K., 2007. Nanoparticle-based oral drug delivery system for an injectable antibiotic –streptomycin,evaluation in a murine tuberculosis model. Chemotherapy 53, 437–441. Sanchez-Silos, R.M., Perez-Giraldo, C., Gomez-Garccia, A.C., 2001. Resistance to vancomycin, LY33328, ciprofloxacin and Trovofloxacin of community acquired and nosocomial strains of Enterococcus faecalis isolated in Badajoz (Spain) with and without high-level resistance to streptomycin and gentamycin. Chemotherapy 47, 415–420. Singh, H., Bhandari, R., Kaur, I.P., 2013. Encapsulation of Rifampicin in a solid lipid nanoparticulate system to limit its degradation and interaction with Isoniazid at acidic pH. Int. J. Pharm. 446, 106–111. Thorne, R.G., Hanson, L.R., Ross, T.M., Tung, D., Frey, W.H., 2008. Delivery of interferon beta to the monkey nervous system following intranasal administration. Neuroscience 152, 785e797. Uner, M., 2006. Preparation, characterization and physico-chemical properties of solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC): their benefits as colloidal drug carriers systems. Pharmazie 1, 375–386.
233
Verma, M.K., Singh, M., kakkar, V., Kaur, I.P., 2012. Applicability of a colorimetric method for evaluating streptomycin sulphate loaded solid lipid nanoparticles. Int. J. Pharm. Pharm. Sci. 4, 50–53. Vyas, T.K., Shahiwala, A., Marathe, S., Misra, A., 2005. Intranasal drug delivery for brain targeting. Curr. Drug Deliv. 2, 164–175. Wermling, D.P., Miller, J.P., Archer, S.M., Manaligod, J.M., Rudy, A.C., 2001. Bioavailability and pharmacokinetics of lorazepam after intranasal, intravenous and intramuscular administration. J. Clin. Pharmacol. 41, 1225–1231. WHO, 2010. Treatment of Tuberculosis: Guidelines for National Programmes. In Vol. WHO/CDS/TB/2003.31. World Health Organization, Geneva. Wilding, I.R., Coupe, A.J., Davis, S.S., 2001. The role of [gamma]-scintigraphy in oral drug delivery. Adv. Drug Deliv. Rev. 46, 103–124. Zensi, A., Begley, D., Pontikis, C., Legros, C., Mihoreanu, L., Wagner, S., Büchel, C., Briesen, H.V., Kreuter, J., 2009. Albumin nanoparticles targeted with Apo E enter the CNS by transcytosis and are delivered to neurones. J. Control. Release 137, 78–86.
