Electronic Supplementary Material (ESI) for RSC Advances.

This journal is © The Royal Society of Chemistry 2015

Supplementary Material:

2.3.1. Quality target product profile

The quality target product profile (QTPP) is defined as “A prospective summary of the quality characteristics of a drug product
that ideally will be achieved to ensure the desired quality, taking into account safety and efficacy of the drug product”. The
target product profile forms the basis of design for development of the product1. Product attributes defining the QTPP for
nanostructured lipid carriers includes particle size, polydispersity index, entrapment efficiency and stabilization of nanoparticles
(ionic or steric hindrance). Justification for selection of attributes and desired target levels for each product attribute have been
tabulated in Table I.

Table I. Quality target product profile of ANLC

Product Attribute Desired Target Justification

Attribute Level
Particle Size (nm) <200 nm Based on the literature report, the particle size <200 nm have potential to
(d=50%) cross blood brain barrier. Since brain is the desired target for action of
asenapine, we have decided to make the nanoparticles in size of below 200
nm2, 3.
Entrapment >60% High drug entrapment permits lower total volume/excipient for administration
Efficiency (% Drug of formulation. This minimizes the undue exposure of excipients to tissues
Loaded) which may occur due to lower entrapment efficiency or high volume dose.
Polydispersity <0.3 Since polydisperse system have greater tendency to aggregation than
Index (PDI) monodisperse system a lower PDI is desired to target the achievement of a
stable nanoparticulate system.
Ionic/steric Stable Since the lipids and surfactants screened for the formulation are non-ionic, the
stabilization Nanosuspension charge on particles was predicted to be near neutral range with some residual
charge from other sources. This was confirmed during the initial trails and
evaluation of formulation characteristics (Zeta Potential: -2 to -5.98 mV). It was
concluded that the stability of nanosuspension will be achieved by steric
hindrance.

2.3.2. Critical material attributes and process parameter

Critical material attribute (CMA) and critical process parameters (CPP) are defined as “A material or process whose variability
has an impact a critical quality attribute and therefore it should be monitored or controlled to ensure desired drug product
quality”. The Critical Quality Attribute (CQA) of drug product and their QTPP were contributed in selection of excipient and
process parameters1. For preparation of ANLC, critical material attributes of drug substance asenapine and excipients including
solid lipid, liquid lipid and surfactant were accessed. Further, two critical process parameters: homogenization speed and
sonication time, were included based on the selected method for preparation of NLC (high shear homogenization and
sonication). The justifications for selection of materials and process parameters have been tabulated in Table II.
Table II. Critical material attributes and critical process characteristics of ANLC.

Drug substance attribute

Drug Substance Justification
BCS Class Class II Asenapine is classified as a BCS Class II compound (low solubility, high
permeability). Low solubility problem may be overcome by nanonization of
particles.
Sublingual 35% The low sublingual bioavailability and high gastro instability of asenapine,
bioavailability makes it a suitable candidate for nanoparticulate lipid drug delivery system.
Oral bioavailability <2% The lipid coat over the particles prevents metabolism.

Excipients attribute
Excipient Justification
Selection of Solid Glyceryl Drug solubility was evaluated in stearic acid, glyceryl monostearate, Compritol
Lipid monostearate 888 ato and Precirol ATO 5. Based on high solubility, Glyceryl monostearate
was selected as solid lipid. (Data not shown)
Selection of Liquid Oleic acid The trial batches were prepared with fixed ratio of liquid lipid (oleic acid,
lipid caprylic/capric triglyceride and propylene glycol dicaprylate/dicaprate) to solid
lipid (GMS). The minimum particle size with sufficiently stable colloidal
dispersion was obtained with oleic acid. (Data not shown)
Selection of Tween-80 Among screened surfactants having brain targeting ability (Tween 80 and
Surfactant Poloxamer 188), it was concluded that Tween 80 stabilized colloidal dispersion
resulted in low particle size and had acceptable stability. (Data not shown)

Critical process parameters

Process Justification
Homogenization 8000-16000 rpm The homogenization speed ranges were selected based on instrument
speed limitation and trial batches. The homogenization speed less than 8000 rpm
leads to large particle size and polydisperse colloidal system. However, the
upper range was set at 16000, since no significant difference in particle size
was observed above 16000 rpm. (Data not shown)
Sonication time 5-15 min The time duration for sonication was selected based on the literature and trial
batches. Moreover, longer duration of sonication was avoided due to leaching
of drug from matrix and possible metal contamination 4. (Data not shown)

Table III. Composition of different batches and their response.

Batch No Composition and process variables Results

OA/ ASM/ Tween-80 HS ST PS (nm) EE (%) PDI

GMS GMS (%w/v) (rpm) (minute)
(w/w) (w/w)
NLC -1 0.15 0.15 1.00 12000.00 10.00 274.65±4.51 75.72±1.83 0.293±0.015
NLC -2 0.10 0.20 1.00 12000.00 10.00 309.75±3.63 69.62±2.52 0.361±0.027
NLC -3 0.15 0.15 1.00 12000.00 10.00 275.63±5.46 74.83±1.58 0.293±0.023
NLC -4 0.15 0.10 1.50 12000.00 10.00 195.51±6.35 59.93±2.62 0.220±0.018
NLC -5 0.10 0.15 0.50 12000.00 10.00 298.46±2.23 67.19±3.23 0.415±0.038
NLC -6 0.15 0.15 1.00 8000.00 5.00 310.98±.3.71 87.22±1.71 0.373±0.022
NLC -7 0.15 0.20 0.50 12000.00 10.00 334.61±4.89 89.78±2.34 0.393±0.027
NLC -8 0.15 0.15 1.00 12000.00 10.00 275.61±2.35 74.14±2.61 0.296±0.011
NLC -9 0.15 0.15 1.00 16000.00 5.00 250.39±2.34 77.56±3.27 0.333±0.029
NLC -10 0.10 0.15 1.00 12000.00 5.00 297.27±5.25 69.72±1.54 0.293±0.021
 NLC -11 0.15 0.10 1.00 12000.00 15.00 195.33±6.38 52.64±1.35 0.196±0.012
NLC -12 0.15 0.20 1.00 12000.00 15.00 245.51±4.63 69.26±2.42 0.250±0.023
NLC -13 0.15 0.15 1.00 12000.00 10.00 278.39±2.36 76.16±2.31 0.295±0.018
NLC -14 0.20 0.15 1.00 8000.00 10.00 274.52±2.02 91.94±2.12 0.257±0.017
NLC -15 0.15 0.15 0.50 8000.00 10.00 316.24±5.62 88.16±2.62 0.396±0.026
NLC -16 0.15 0.10 1.00 16000.00 10.00 215.21±4.66 60.35±1.14 0.248±0.021
NLC -17 0.20 0.20 1.00 12000.00 10.00 305.14±7.63 92.56±1.65 0.255±0.024
NLC -18 0.15 0.15 1.50 12000.00 5.00 230.15±6.42 76.78±2.57 0.303±0.014
NLC -19 0.15 0.15 1.50 8000.00 10.00 223.62±5.72 69.71±2.67 0.277±0.026
NLC -20 0.15 0.15 1.50 16000.00 10.00 165.62±6.36 59.74±3.60 0.237±0.012
NLC -21 0.10 0.10 1.00 12000.00 10.00 262.32±4.68 52.42±2.46 0.310±0.028
NLC -22 0.10 0.15 1.00 12000.00 15.00 211.35±7.56 74.97±3.25 0.271±0.022
NLC -23 0.15 0.15 1.00 12000.00 10.00 277.34±8.34 74.26±2.61 0.295±0.013
NLC -24 0.15 0.10 1.00 8000.00 10.00 275.62±7.35 70.62±2.52 0.290±0.024
NLC -25 0.20 0.15 0.50 12000.00 10.00 291.25±7.25 92.27±1.75 0.306±0.031
NLC -26 0.15 0.20 1.50 12000.00 10.00 245.36±4.40 76.24±2.74 0.275±0.023
NLC -27 0.20 0.15 1.50 12000.00 10.00 198.53±3.83 86.18±2.96 0.184±0.013
NLC -28 0.15 0.20 1.00 12000.00 5.00 331.32±2.52 91.36±1.71 0.374±0.032
NLC -29 0.15 0.10 0.50 12000.00 10.00 284.21±3.61 75.82±4.38 0.343±0.012
NLC -30 0.15 0.20 1.00 16000.00 10.00 265.76±7.34 77.93±2.23 0.305±0.022
NLC -31 0.15 0.15 1.00 12000.00 10.00 273.46±6.32 72.61±3.21 0.297±0.021
NLC -32 0.20 0.10 1.00 12000.00 10.00 251.25±9.46 87.13±3.67 0.203±0.024
NLC -33 0.15 0.15 1.00 8000.00 15.00 224.97±6.30 62.28±2.36 0.252±0.025
NLC -34 0.15 0.20 1.00 8000.00 10.00 325.63±4.75 87.86±3.83 0.345±0.032
NLC -35 0.20 0.15 1.00 12000.00 5.00 288.26±4.72 94.36±1.32 0.282±0.032
NLC -36 0.15 0.10 1.00 12000.00 5.00 280.45±9.43 77.15±4.86 0.317±0.031
NLC -37 0.10 0.15 1.50 12000.00 10.00 210.15±5.27 51.72±3.98 0.294±0.026
NLC -38 0.10 0.15 1.00 8000.00 10.00 290.52±8.21 62.78±4.92 0.368±0.014
NLC -39 0.15 0.15 0.50 12000.00 15.00 236.26±3.67 67.37±2.71 0.301±0.030
NLC -40 0.20 0.15 1.00 16000.00 10.00 226.86±4.44 86.62±3.72 0.213±0.021
NLC -41 0.15 0.15 1.50 12000.00 15.00 147.78±2.35 51.28±4.22 0.182±0.012
NLC -42 0.10 0.15 1.00 16000.00 10.00 230.24±3.56 51.23±3.14 0.324±0.019
NLC -43 0.15 0.15 1.00 16000.00 15.00 165.26±2.46 52.67±2.84 0.211±0.015
NLC -44 0.20 0.15 1.00 12000.00 15.00 201.62±4.72 79.16±2.92 0.163±0.012
NLC -45 0.15 0.15 0.50 12000.00 5.00 322.62±3.47 89.45±3.17 0.423±0.038
NLC -46 0.15 0.15 0.50 16000.00 10.00 253.25±3.78 75.26±2.29 0.353±0.033
HS: Homogenization speed, ST: Sonication time, PS: Particle size, EE: Entrapment efficiency, PDI: Polydispersity index, mean±SD,
n=3.

Reference:
1. ICH Q8(R2) Pharmaceutical Development (2005) http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/
Guidelines/Quality/Q8_R1/Step4/Q8_R2_Guideline.pdf ). [Accessed on May 21, 2015].
2. S. Martins, I. Tho, I. Reimold, G. Fricker, E. Souto, D. Ferreira and M. Brandl, International Journal of Pharmaceutics,
2012, 439, 49-62.
3. L. Kozlovskaya, M. Abou-Kaoud and D. Stepensky, Journal of controlled release, 2014, 189, 133-140.
4. J. N. Betts, M. G. Johnson, P. T. Rygiewicz, G. A. King and C. P. Andersen, Environmental toxicology and chemistry /
SETAC, 2013, 32, 889-893.