Biomimetic Solid Lipid Nanoparticles of Sophorolipids Designed for

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B: Fluid Interfaces, Colloids, Polymers, Soft Matter, Surfactants, and Glassy Materials

Biomimetic Solid Lipid Nanoparticles of Sophorolipid Designed for Anti-Leprosy Drugs Rohini Kanwar, Michael Gradzielski, and Surinder K. Mehta J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b03081 • Publication Date (Web): 06 Jun 2018 Downloaded from http://pubs.acs.org on June 6, 2018

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The Journal of Physical Chemistry

Biomimetic Solid Lipid Nanoparticles of Sophorolipid Designed for AntiLeprosy Drugs Rohini Kanwar,1 Michael Gradzielski,2* and S.K. Mehta1* 1

Department of Chemistry and Centre for Advanced Studies in Chemistry, Panjab University,

Chandigarh-160014, India 2

Stranski Laboratorium für Physikalische und Theoretische Chemie, Institut für Chemie,

Technische Universität Berlin, D-10623 Berlin, Germany

_________________________________________________________________________ Corresponding authors: S. K. Mehta and Michael Gradzielski Tel: +91-172-2534423 Fax: +91-172-2545074 Email addresses: [email protected]; [email protected]

1 ACS Paragon Plus Environment

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ABSTRACT The objective of the present work was to develop solid lipid nanoparticles (SLNs) as drug encapsulating structures by solvent injection method. In this report, for the first time the inherent potential of lactonic sophorolipid (glycolipid) was exploited to formulate SLNs. A range of different Pluronic co-polymers were screened by dynamic and static light scattering with the aim of obtaining most stable SLNs. In order to comprehend the structure of the SLNs, techniques such as transmission electron microscopy (TEM), differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FT-IR) and X-ray diffraction (XRD) were employed. A clear correlation between the type of Pluronic and size and stability of the SLN could be drawn. The vector properties of the formed SLNs were assessed for both the encapsulated hydrophobic drugs- rifampicin and dapsone. To elucidate the transport mechanism of drug release, kinetic modelling was carried out on the drug release profiles. The promising results of sophorolipid based SLNs has actually established a new arena beneath the significantly developed field of SLNs.


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1. INTRODUCTION Solid lipid nanoparticles (SLNs) are ubiquitously investigated encapsulation cargos for hydrophobic entities such as drugs,1 additives or cosmetics.2 They are composed of a core of physiological lipids (that are solid at both room and body temperature) stabilized by a surfactant solution. SLNs are basically the alternative version of emulsions in which the liquid state of the lipid (oil) has been replaced with the solid state (similar to polymeric nanoparticles). They are fully crystallized where drugs are accommodated within the lipid matrix to shield them against chemical degradation under harsh conditions, by decreasing the mobility of drugs in the solid state which thereby allows for slow controlled release.3 They encompass various advantages like ease of preparation, high entrapment efficiency, excellent biocompatibility, no biotoxicity of the carrier, stability against coalescence, controlled drug release and targeting etc.4 Sophorolipid is one of the most promising biosurfactants that combines green chemistry with low carbon footprint (typically lower unwanted side products or environmental snags associated with conventional synthetic surfactants).5 As a surfactant, it combines a number of properties like stability over a wide temperature range, pH and salinity.6 Its low-foaming,7 resistance to water hardness, and the synergism between acidic and lactonic forms of sophorolipid increases its surfactant activities,8 while biodegradability and good surface activity (emulsification behavior),9 makes this class of glycolipid superior to many existing synthetic surfactants. Apart from the above mentioned positive attributes, they can be produced in large quantities10 based on renewable resources, agro-industrial by-products11 and residues,12 and easy and simplified product recovery.13 These immanent traits of sophorolipids have made these species an active



ingredient for divergent cellular mechanisms owing to their bio-derived origin, therapeutic potential for antiviral, antimicrobial and anti-inflammatory treatments.14 Sophorolipid has been solely exploited as promising biosurfactant (for the fabrication of niosomes, liposomes, microemulsion, etc.) however, till now, it has not been deployed as the lipidic source for the generation of lipid nanoparticles for therapeutic concerns.15,16 The lacunae in the exploration of sophorolipid as the lipidic source led to development of sophorolipid based SLN so that inherent property of this lipid is exploited. Lactonic sophorolipid is selected over acidic sophorolipid due to its better antimicrobial, antiviral, antifungal, anti-inflammatory and anticancer activities, as evidenced by Cortes-Sanchez et al.17 Rifampicin and dapsone being broad-spectrum antibiotics are suitable for the treatment of bacterial infections of Mycobacterium leprae. According to biopharmaceutical classification (BCS), these drugs belong to class II type drugs (hydrophobic) where solubility problem leads to less bioavailability issue and low therapeutic index.18,19 Therefore, SLNs are fabricated, in order to address the issues like solubility, stability and bioavailability of these drugs efficiently. Figure 1 shows the chemical structure of the lipid, emulsifier and leprosy drugs. In the present study, SLNs have been fabricated by coating with nonionic polymeric surfactant of the type poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethylene oxide) copolymers (PEO-PPO-PEO)(Pluronics) using the solvent injection method with the ultimate aim of administration of anti-leprosy drugs. Pluronics are non-ionic triblock copolymers composed of a hydrophobic PPO central block linked to hydrophilic PEO end blocks. These were selected owing to the positive attributes i.e. flexibility with respect to molecular architecture, tunable size and hydrophilic-hydrophobic balance (HLB), approved medical usage as drug or gene delivery vehicles etc. The effect of surfactant type and concentration on the size


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and stability of the developed SLN was investigated for a period of 28 days with the help of dynamic and static light scattering (DLS, SLS). The in depth physicochemical characterization of the optimized SLN formulations was done with transmission electron microscopy (TEM), differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FT-IR) and Xray diffraction (XRD). Finally, the vector properties of SLN were assessed for both the encapsulated hydrophobic drugs- rifampicin and dapsone. CH3 H3 C

O

O O

O OH OH

H 3C

O

O O OH

CH3

O O

O OH

H

OH

O PEO

O

x

PPO

y

PEO

O

1',4''-Sophorolactone 6',6''-diacetate (Sophorolipid)

Pluronics

HO O

OH O OH OH

O

O

NH

O S

H3CO N

O

N

O

OH

H2N

N

NH2

O

Dapsone

Rifampicin

Fig. 1. Chemical structure of the lipid, emulsifier and leprosy drugs.

2. MATERIALS AND METHODS


z


Materials. 1′,4″-Sophorolactone 6′,6″-diacetate (99%, produced from fermentation of sugar by yeasts) was taken from Cayman Chemicals. Rifampicin (≥ 97%) and dialysis tubing (molecular weight cut off 12-14 kDa) was purchased from Sigma Aldrich. Dapsone (≥ 97%) was obtained from Himedia. Pluronics P65, F68, P84, P123 and F127 were obtained as a gift from BASF. Ethanol (99.9%) was taken from Changshu Yuang Chemicals. Deionized water with resistance of 18 mΩ was utilized for all the experiments. All the compounds were employed as received. Optimization of SLNs. Fabrication of SLNs was carried out by solvent injection method.20 Briefly, lipidic phase was made by dissolving sophorolipid (50 mg) in 1.0 mL of ethanol and then the mixture was rapidly injected through an injection needle into a stirred (1000 rpm) aqueous surfactant solution consisting of 10 mL of deionized water containing different concentrations (0.2 to 4%) of surfactants (Pluronic P65, F68, P84, P123, and F127) at 60 °C (Fig. S1). The obtained dispersion was stirred at 40 °C for 30 mins so that the lipid crystallizes due to the diffusion of organic solvent from the organic phase to the aqueous phase followed by evaporation of ethanol. Later, the mixture was dispersed into ice-cold water (at 4 °C) drop wise in 1:1 volume ratio followed by stirring for 30 mins at 1000 rpm and centrifugation for 15 mins


Fig. 2. Schematic representation of SLNs formation.

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at 10,000 rpm. The cooling step actually promoted the formation of SLNs. The resulting nanoparticles suspension was then lyophilized/freeze-dried and kept under storage for future experiments. Figure 2 shows the schematic representation of SLNs formation. Physicochemical Characteristics of SLNs. The hydrodynamic radius and radius of gyration were determined by dynamic light scattering (DLS) and static light scattering (SLS) experiments using an ALV/CGS-3 instrument (equipped with an ALV5000/E multiple-τ correlator and ALVSP 125 goniometer) at TU Berlin. A broad range of scattering angles from 40° to 130° was chosen to get information at different magnitudes of the scattering vector q. Microscopic analysis of the fabricated SLNs was done by using transmission electron microscopy (FEI Tecnai G2 20 S-TWIN TEM instrument). The physical state on the lipid was determined by doing differential scanning calorimetry (DSC) measurements at DSC-Q20 (TA Instrument, USA). Heating curves for the SLNs, and its corresponding excipients were recorded with a scan rate of 10 °C/min from 0 °C to 150 °C. Each sample was run three times to check out the reproducibility of the result. Fourier transform infrared spectroscopy (Thermo Scientific-Nicolet iS50 FT-IR) was employed to understand the structural make up of SLNs. A PANalytical Xpert pro X-ray diffractometer (XRD) equipped with Cu-Kα radiation source (λ = 1.541 Å) was utilized to record the diffraction pattern of the excipients and SLNs over the 2θ range from 5° to 100°. Physical Stability. The physical stability of the SLNs dispersion was assessed by examining the changes in the hydrodynamic radius, radius of gyration and physical appearance during storage at room temperature protected from the light. Drug Loaded SLNs (DL-SLNs). The leprosy drugs- rifampicin and dapsone (4 mg) were loaded into the lipidic phase of SLNs. The UV absorbance was noted down by a JASCO V530 spectrophotometer in the range of 200-600 nm and the maximal absorbance was obtained at



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475 nm for rifampicin and 295 nm for dapsone. The stability of pure as well as DL-SLNs was tested by carrying out centrifugation at 10,000 rpm for 15 min to precipitate out the particle aggregates and excess drug. Entrapment Efficiency (EE) and Drug Loading Capacity (LC). Dialysis membrane method was employed to determine the amount of drug entrapped in the SLNs and its corresponding loading capacity. 2 mL dispersion of DL-SLNs filled in dialysis bag was immersed into the 50 mL phosphate buffer (pH=7.4) at 25 ± 0.1 °C (Lauda Ecoline RE320 thermostat) maintained at 100 rpm stirring and monitored for 5 h to measure the absorbance of the dialysate. The entrapment efficiency (EE) and loading capacity (LC) of DL-SLNs were calculated according to the following equations:21 EE (%, w/w) = ቂ

ୟ୫୭୳୬୲ ୭୤ ୢ୰୳୥ ୧୬ ୲୦ୣ ୤୭୰୫୳୪ୟ୲୧୭୬ିୟ୫୭୳୬୲ ୭୤ ୤୰ୣୣ ୢ୰୳୥

LC (%, w/w) = ቂ

ୟ୫୭୳୬୲ ୭୤ ୢ୰୳୥ ୧୬ ୲୦ୣ ୤୭୰୫୳୪ୟ୲୧୭୬

ቃ × 100

ୟ୫୭୳୬୲ ୭୤ ୢ୰୳୥ ୧୬ ୲୦ୣ ୤୭୰୫୳୪ୟ୲୧୭୬ିୟ୫୭୳୬୲ ୭୤ ୤୰ୣୣ ୢ୰୳୥ ୵ୣ୧୥୦୲ ୭୤ ୪୧୮୧ୢ

ቃ × 100

(1) (2)

To verify the authenticity of results, the experiments were performed in triplicate. In-vitro Release Study of DL-SLNs. Similar to the method described for calculating EE, the dialysis bag diffusion technique was utilized. The difference lies in the temperature of experiment i.e. maintained at 37 ± 0.1 °C and, at predetermined time intervals, aliquots of dialysate (2 mL) were collected and replaced immediately with equal volumes of fresh media for 5 h.22 To check the reproducibility of results, release studies were performed in triplicate (SD of ± 0.2%). Kinetic Modelling. Different mathematical models [Eqs. S1-S8, given in the Supplementary Information] were taken into account to understand the kinetic behavior and release mechanism of drugs from DL-SLNs and the corresponding linear regression lines were compared to determine the order of release kinetics.23,24 8 ACS Paragon Plus Environment

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3. RESULTS AND DISCUSSION Optimization of SLNs. The selection of emulsifier and its concentration is an important parameter that accounts for the quality and long term physical stability of SLN dispersion. In the present study, the five different polymers, Pluronic F68, F127, P123, P65, and P84 (Table S1) with different PEO content (%) were employed while keeping the amount of lipid constant (50 mg). First, all the polymers were employed to formulate SLNs, in which the amount of co-polymer was kept constant at 1.5 (w/v%). When DLS and SLS measurements were conducted to obtain the most stable SLNs, it was observed that the particle size is significantly influenced by the usage of the different surfactants. Among the different Pluronics tested, SLNs prepared using Pluronic F127 and P123 were smallest (~10 nm) and SLNs prepared using F68 were largest (~200 nm). The apparent hydrodynamic radius (Rh) decreased as follows: F68 > P65 > P84 > F127 > P123 (Fig. 3a). In the case of Pluronic F127 and P123, the formed smaller sized particles are simply micelles (of ~10 nm size). Although they have different PEO content, the PPO content is almost the same, which is why they behave similarly. F68 and P65 are also relatable as they have same PPO content but the higher PEO content i.e. 80% in the case of F68 accounts for its bigger particle size than P65 which have only 50% PEO content. Therefore, it can be inferred that as the PEO content increases, the particle size increases in the following order: P84 < P65 < F68. Further, on monitoring SLNs as a function of ageing time (for a month), F68 exhibited the least variation in Rh in comparison to P65 and P84. This can again be explained on the basis of a better steric stabilization capability of F68 due to the presence of higher percentage of PEO (%) 9 ACS Paragon Plus Environment


content than others, as apparently the longer EO head group increases the colloidal stability. For further experiments P65 and P84 based SLNs were excluded (due to stability issues) and F68, F127 and P123 based SLNs were chosen, due to their better colloidal stabilization of the larger particles (SLNs). Table 1 Formulation Design of Slns with Characteristic Physicochemical Parameters of Freshly Prepared Samples Given are the diffusion coefficient D, hydrodynamic radius Rh, polydispersity index PdI, and radius of gyration Rg.


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Formulations

SLN 1 SLN 2 SLN 3 SLN 4 SLN 5 SLN 6 SLN 7 SLN 8 SLN 9 SLN 10 SLN 11 SLN 12 SLN 13 SLN 14

Type of Surfactant

Surfactant concentration (w/v%) 0.5 1.0 Pluronic F68 1.5 2.0 2.5 0.50 0.75 Pluronic F127 1.0 1.5 2.0 2.5 Pluronic P123 1.5 Pluronic P65 1.5 Pluronic P84 1.5

Diffusion Coefficient in µm2/s 0.40 0.85 1.17 1.45 1.53 1.01 1.18 20.09 22.62 19.95 21.60 25.59 1.67 1.76


Rh (nm) from DLS

PdI

Rs (nm) from SLS

614 287 210 169 159 242 207 12.2 10.8 12.3 11.3 9.58 147 139

0.28 0.59 0.19 0.17 0.18 0.29 0.28 0.25 0.15 0.14 0.16 0.12 0.17 0.35

164 149 144 138 135 171 136 135 151


a)

b)

c)

d)

f)

e)

SLN-3 SLN-7

-3.0

ln I (q) (nm-1)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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-3.5

-4.0

-4.5

0.0000

0.0002

0.0004 q2 (nm-2)

0.0006

Fig. 3. Apparent hydrodynamic radius of SLNs suspension (obtained from DLS) as a function of ageing time of a) Pluronics b) F68 (w/w%) c) F127 (w/w%) ; the corresponding static radius (obtained from SLS) for varied concentration of d) F68 e) F127; and f) plot of ln (I) vs. q2 obtained from SLS (after 12

ACS Paragon Plus Environment Guinier approximation) for selected SLN-3 and SLN-7 formulations.

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In another part of the study, the effect of surfactant concentration on SLNs was evaluated with the help of DLS and SLS experiments. The concentration window starting from 0.5 to 2.5 (w/v%) was selected after ensuring that the value of critical micellar concentration (cmc) lies below 0.5 (w/v%) for all the Pluronics (Table S1). In the case of F68, a regular decrease in size from 0.5 to 2.5 (w/v%) was observed, as shown in Table 1 (Fig. 3b and 3d). With increasing surfactant concentration, the larger interface that can be stabilized facilitates the breakdown of lipid drops into smaller size and reduces the particle size.25-27 After 1 month storage of samples, the SLNs with low surfactant concentration of 0.5 (w/v%) aggregated, as not enough surfactant was present to cover the tiny lipid droplets surface effectively, i.e., and as a result bigger particles were formed and settled down due to insufficient repulsion among particles. In contrast, at higher surfactant concentration, the nanoparticles remained stable and their coalescence was prevented. However, above 1.5 (w/v%) concentration of surfactant, the stability and particle size did not improve further. Therefore, 1.5 (w/v%) F68 was selected to formulate the most stable SLNs. On the other hand, F127 is also extensively utilized in fabricating SLNs owing to its approved medical usage.28 Therefore, a concentration window for F127 was employed from 0.5 to 2.5 (w/v%). It was inferred from particle size measurements that instead of micelles, SLNs with Rh= 242 nm at 0.5 (w/v%) and 207 nm at 0.75 (w/v%) can be prepared (at concentration below 1.0 (w/v%)). Again, with passaging time, samples with 0.5 (w/v%) F127 aggregated to much larger structures, due to a too low amount of surfactant required to cover the surface of lipid particles. However, 0.75 (w/v%) remained stable even after a month storage (Fig. 3c and 3e).



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In general, to formulate stable o/w emulsions, a hydrophilic-lipophilic balance (HLB) greater than value 12 is considered to be ideal, where, on one side, F68 (HLB=29) and F127 (HLB=22) act as good emulsifiers due to higher HLB, and, P123 having HLB=8, is considered unsuitable to prepare SLNs. Therefore, Pluronic F68 and F127 have been chosen as the appropriate surfactants for preparing SLNs (SLN-3 and SLN-7, respectively) owing to their better emulsification capability and lesser toxicity potential. The selected SLNs are also expected to increase the bioavailability of the encapsulated drugs based on the smaller sizes of SLNs.18 Figure 3f shows the plot of natural logarithm of the light scattering intensity (I) versus the square of scattering vector (q) for SLN-3 and SLN-7 systems according to the Guinier approximation: lim ‫ ܫ‬ሺ‫ݍ‬ሻ = ‫ ܫ‬ሺ0ሻ. ݁‫ ݌ݔ‬൬−

௡→௤

௤ మ .ோ೒ మ ଷ

൰

(3)

From the value of radius of gyration around the center of mass of the particle (Rg), the static radius of spherical shaped particles (Rs) were calculated by the relationship: ܴ௦ = ට5ൗ3 ܴ௚

(4)

Here, in this case, radius of spherical particles was found to be 144 nm and 136 nm for SLN3 and SLN-7, respectively. On comparing the results of DLS and SLS measurements, it could be inferred that the more accurate information about the size distribution is attained from SLS data, as problem of Rh dependence on scattering angle (when such large sized nanoparticles are present) encountered in analyzing DLS data is avoided. Physicochemical Characterization. TEM examination of lyophilized samples (SLN-3 and SLN-7 formulations) also supported the SLS results and revealed that spherical shaped nanoparticles are present in both SLNs formulation (Fig. 4). Very small sized SLNs were 14 ACS Paragon Plus Environment

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noticeable under TEM ranging from 100 to 150 nm sized nanoparticles for both the systems. Additionally, it showed that lipid core is entrapped and stabilized inside the non-ionic poloxamer shell. No evident sign of aggregation was found. 5

5

a)

b)

4

Counts

4

Counts

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3

3

2

2

1

1

0

100

110

120

130

140

150

Particle Size (nm)

0

110

120

130

Particle Size (nm)

Fig. 4. TEM images of a) SLN-3 and b) SLN-7 formulation (i.e., scale bar = 100 nm) with insets showing their respective histograms of particle size distribution. DSC analysis was employed to gain an insight of the crystalline structure and melting behavior of the lipid matrix under the influence of stabilizer coating with poloxamers. Figure 5a gives DSC thermographs of pure excipients and lipid nanoparticles whereas Table 2 shows the corresponding parameters such as onset temperature, melting point, and enthalpy values. The characteristic peak of pure sophorolipid showed a melting point at 75.68 °C with enthalpy change (∆H) of 28.35 J/g. The pure poloxamers exhibited sharp crystalline peaks with higher enthalpy values. The ∆H decrease for SLNs in comparison to pure surfactants (Pluronic F68 and F127) referred towards the lesser crystallinity of nanoparticles than poloxamers, thereby, indicates the entrapment of the lipid inside the emulsifier shell. Therefore, a clear distinction between the pure lipid and its corresponding nanoparticles can be made as the poloxamers make a coating surrounding the nanoparticles and makes it more crystalline. The 15 ACS Paragon Plus Environment


melting point depression and sharper heating peak of the SLNs with respect to the bulk lipid can be attributed to the Kelvin effect (and explained by the Thomas equation) and is due to the formation of nanosized particles, its low dimensions, particularly large surface-to-volume ratio/high specific surface area and presence of surfactant.29,30 The presence of a single melting peak or melting enthalpy in case of SLNs, clearly excludes the possibility for existence of supercooled melt. As the onset and melting temperature of the fabricated SLN-3 and SLN-7 is higher than 40 °C, ensures the formation of SLNs at body temperature and also, makes it a suitable candidate for topical route of administration of lipid nanoparticles.31 To comprehend the structural arrangement, FTIR spectra was recorded from 4000 to 500 cm1

range of wavelength. Figure 5b shows the FTIR spectra for all the excipients and SLNs system.

When the fabricated SLNs formulations were compared with the pure sophorolipid, it was observed that the coupled peak of asymmetrical stretching at 2929 cm-1 and symmetrical stretching at 2859 cm−1 for methylene (CH2) group and the peaks at 1368, 1232, 1115, 1062 and 837 cm−1 vanished. Only an absorption band at 1740 cm−1 contributing to the C=O stretching of lactone ester in sophorolipid remained persistent. The entire spectra of SLNs overlaps with the pure surfactants (poloxamers). Further, the powder XRD patterns of pure excipients and lyophilized SLN-3 and SLN-7 are shown in Fig. 5c. XRD curves showed that pure lipid mainly exists in an amorphous state, whereas pure surfactants and formulated SLNs are present in crystalline state. The change in relative intensity ratio of the crystalline peaks present in SLNs (compared to pure surfactants) and the change in d-spacing of pure surfactants after lipid encapsulation, simply reflects the structural rearrangement of the poloxamers that can only happen if the lipid disturbs the


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crystalline structure of poloxamers and gets accommodated in the structure or modification of crystalline nature of the material. Table 2 shows the utilized parameters and the obtained size values from XRD using the Debye Scherrer equation (5): ‫=ܦ‬

௄ఒ

(5)

ఉ ஼௢௦ఏ

where, K is the shape factor (0.94), λ is the X-ray wavelength, β is the full width half maximum

112

-0.32

a)

105

LIPID

b) LIPID

98 91

-0.64

84

PLURONIC F68

105

PLURONIC F68

84

-2.4

63 42

-4.8

% Reflectance

Endothermic heat flow (W/g)

0.0

PLURONIC F127

0.0 -1.8 -3.6

SLN-3

0.00

21 104

PLURONIC F127

91 78 65 52 105

SLN-3

84

-0.98

63 42

-1.96

21

SLN-7

SLN-7

105

-0.48

84 63

-0.96

42 21

-1.44

30

60

90

4000

120

3500

3000

2000

2500

2000

1500

1000

500

Wavenumbers (cm-1)

Temperature (οC)

c)

1000 LIPID 12000 8000

Intensity (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


4000 PLURONIC F68 8000

4000 PLURONIC F127 6000 4000 2000 SLN-3 3600 2400 1200

SLN -7

20

40

60

80

2θ

Fig. 5. a) DSC thermographs (T = 0 to 150 °C) b) IR spectra (T = 25 °C) and c) XRD patterns of pure lipid, Pluronic F68, F127, SLN-3 and SLN-7 (T = 25 °C). 17 ACS Paragon Plus Environment


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intensity of most abundant peak, θ is the Bragg’s angle in radians and D is the mean size (nm) of the particle. Table 2 DSC and XRD Parameters of Pure Excipients and Lyophilized SLN-3 and SLN-7 Formulations Given are the angle of the peak position 2θ, the resulting spacing d, full width half maximum intensity β, and crystallite size D (as obtained by applying eq. 5)/ Sample Melting Enthalpy D Average Onset (°C) 2θ (°) d (nm) β (radian) -2 (×10 ) (nm) ‘D’ (nm) point (°C) ∆H (J/g) Sophorolipid 66.67 75.68 28.35 Amorphous Pluronic F68

53.21

55.42

139.8

Pluronic F127 SLN-3

52.64

55.57

112.1

45.03

51.06

76.52

SLN-7

42.09

51.13

53.67

19.09 23.41 19.21 23.39 19.38 23.55

4.65 3.80 4.62 3.80 4.58 3.77

0.38 1.40 0.41 1.28 0.64 1.11

38.7 10.6 35.9 11.5 22.9 13.3

24.6

19.43 23.59

4.57 3.77

0.64 1.52

22.9 9.7

16.3


23.7 18.1

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Drug Loaded SLNs (DL-SLNs). The primary objective behind developing these biocompatible DL-SLNs was to overcome the problem of poor bioavailability and low solubility associated with the two anti-leprosy drugs i.e. rifampicin and dapsone. When both the drugs belonging to class II drugs (according to BCS) are loaded into the SLNs, they behave as class I drugs, as the solubility and permeability of the drugs increases tremendously which thereby increases the bioavailability of the drugs. Hence, to get an idea of in-vivo performance of DLSLNs, the vector properties were evaluated. Entrapment Efficiency (EE) and Drug Loading Capacity (LC). The most important property for pharmaceutical usage of drug delivery vehicle resides in encapsulating sufficient

Entrapment efficiency (EE, %)

100

a)

b)

SLN-3 SLN-7

99

0.8

98 0.4

97 96

0.0

95 Rifampicin

Dapsone

Rifampicin

Loading Capacity (LC, %)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Dapsone

Drug

Fig. 6. a) Entrapment efficiency (EE, %) and b) loading capacity (LC, %) for rifampicin and dapsone from SLN-3 and SLN-7. amount of desired therapeutic molecules. For both the drugs, EE was determined by UV-vis spectroscopy using eq. 2. It was found out to be almost same for SLN-3 and SLN-7 for both drugs, but was significantly higher as compared to other reported systems.18,32,33 For rifampicin, the EE (%) obtained was 98.6 ± 0.2 and 98.8 ± 0.2, for SLN-3 and SLN-7 formulations whereas



for dapsone, the values were of 96.8 ± 0.2, and 96.9 ± 0.2 for SLN-3 and SLN-7 formulations, respectively (Table S2 and Fig. 6a). Another significant aspect is the LC, which resolves the problems related to multi-drug dosage. LC was calculated with respect to sophorolipid. In contrast to EE, LC was different for SLN-3 and SLN-7 for both the drugs as shown in Table S2 and Fig. 6b. The high value of EE gives an indication towards the efficient loading and retention of the drug molecules at the surface or within the matrix of SLNs at higher surfactant concentration. However, the EE for both the drugs has been found to be almost same, irrespective of the different structure of drugs. Therefore, it can be anticipated that it is the inner core of the delivery vehicle (hydrophobic core) and the type of interaction between the drug and the nano vehicle which decide the capacity of the vehicle to encapsulate the drug in it. Even the high drug loading can be attributed to the coating of Pluronic F68 and F127 owing to the presence of PPO hydrophobic chains. In-vitro Release of DL-SLNs. Figure 7 depicts the drug release profiles from SLN-3 and

a)

b)

Cumulative Release wt (%)

70

350 300

60 50

250

40

200

30

150 Rifampicin loaded SLN 3 Dapsone loaded SLN 3 Rifampicin loaded SLN 7 Dapsone loaded SLN 7

20 10

100 50

0

0 0

50

100

150

200

250

300

0

50

100

150

200

250

300

Time (mins) Fig. 7. In-vitro drug release profiles for rifampicin and dapsone from SLN-3 and SLN-7 in 20 terms of cumulative a) release wt (%) and b) concentration of drug (µg/mL) for 5 hrs with SD ACS Paragon Plus Environment

(n=3) for all the 25 data points in each case.

Concentration (µg/mL)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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SLN-7 formulations as determined by dialysis method. The difference in the release profiles for both the drugs simply indicate the difference in interaction between the drug and the lipid matrix. However, for different SLN formulation, the pattern of release was similar but SLN-7 showed comparatively higher release than SLN-3 formulation due to comparatively smaller size which in turn decreases the concentration gradients and mass transport rates. It also depicts the influence of different type of emulsifiers on the release rate. The amount of rifampicin released from SLN3 and SLN-7, systems was 46.09% (202.80 µg/mL) and 48.44% (213.13 µg/mL), respectively. After normalizing these values by the surface area of SLNs (by simply dividing the amount of drug released with surface area of SLNs), 0.774 ×1012 and 0.913×1012 g L-1 m2 values are obtained for SLN-3 and SLN-7 systems, respectively, where a good correlation with the size of the nanoparticles is seen. In the case of dapsone, the amount released from SLN-3 and SLN-7 systems was 71.02% (312.47 µg/mL) and 73.06% (321.48 µg/mL), respectively (Table S2). The obtained normalized values were 1.19×1012 and 1.38×1012 g L-1 m2 for SLN-3 and SLN-7 systems, respectively, well correlated with the size of the nanoparticles. The rate of drug release has been found higher than the previously reported18,33 release rates where the released concentration lies well in the therapeutic window and so it is expected to be


Fig. 8. Pictorial representation of two step drug release profile from SLN.


active leprosy ailment with a sustained and prolonged release. The drug release profile from both the SLNs showcases a biphasic pattern with initial burst release followed by a prolonged release. Figure 8 shows the pictorial representation of two-step drug release describing the distribution of drug in the matrix and on the surface of SLNs. The initial fast release of the incorporated drug might result from diffusion from matrix erosion caused by hydrolytic degradation or external particle surface. The subsequent prolonged release might result from the liberation of drug from the lipid core through diffusion and dissolution.34 Kinetic Behavior. Applying Eqs. S1-S8 (given in the Supplementary Information) onto the results obtained from release studies, mode of drug release from SLNs was estimated.23,24 Based on the obtained values of rate constants and regression correlations (Table S3), the best suited model for both the drugs from the SLNs was first order exponential kinetics. The residual for exponential decay was found to be the smallest with no systematic changes, in comparison to rest of the models (Fig. S1). Therefore, it can be inferred that the release kinetics follows a simple first-order law, where the rate of drug release is concentration dependent. In addition, the KP model was employed to comprehend the drug release mechanism in terms of the diffusion exponent (n) and constant (kp) which depicts the release mechanism and, structural and geometrical characteristics of the release device, respectively. For release of rifampicin, both systems show a transport mechanism of anomalous (non-Fickian) diffusion release, and for dapsone, Fickian diffusion driven release. The Fickian driven process indicates that it involves diffusion controlled efflux (due to concentration gradient) whereas, the nonFickian diffusion mechanism infers that the diffusion controlled efflux is coupled with the erosion mechanism.


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4. CONCLUSIONS In this report, a solvent based technique has been employed to generate SLNs where lactonic sophorolipid is dissolved in the organic water-miscible solvent and further dispersed in poloxamer solution. Pluronics-F68 and F127 were found to be the most suitable surfactants for preparing SLNs due to their optimal size stability and lower toxicity potential. The formation of SLNs was confirmed by TEM, DSC, FTIR and XRD studies. Exhibiting a high entrapment efficiency and loading capacity for both the drugs-rifampicin and dapsone, the fabricated SLNs have become of paramount importance. The released drug concentration remained well in the therapeutic concentration window despite of high release rate. The kinetic modelling showed that the transport mechanism of drug release for rifampicin is non-Fickian and for dapsone, it is Fickian driven process. Owing to the easy preparation, biocompatibility, high entrapment efficiency, sustained release and increased bioavailability etc. positive features of both the systems, they have become a lucrative choice for further studies. The unexemplified results of the developed SLNs has provided a new direction to the SLNs for encapsulation of various other hydrophobic drugs.

Acknowledgements Rohini Kanwar is thankful to CSIR and DAAD for fellowship. Surinder Kumar Mehta thanks the PURSE-II Grant for financial assistance.

Supporting Information Available: Kinetic equations; Residual graphs obtained after kinetic modelling; Properties of Pluronics; Entrapment efficiency, loading capacity and in-vitro drug



release parameters after 5 hrs for SLNs; and Rate constant and regression coefficient values obtained after kinetic modelling. Supplementary data associated with this article can be found in the online version, at http://pubs.acs.org.


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TOC GRAPHIC



ACS Paragon Plus Environment

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Biomimetic Solid Lipid Nanoparticles of Sophorolipids Designed for

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