Figure S1 Proposed reaction scheme for synthesis of mannosylated chitosan (Mnos-CS)
Figure S2Representative FTIR spectra of mannosylated chitosan (Mnos-CS) and chitosan (CS)
Lyophlization of nanocapsules
The 2 ml of nanocapsule suspension was filled in 5 ml glass vials. The cryoprotectants i.e. dextrose,e sucrose, trehalose and mannitol were added in vials. The nanocapsule suspension was fast frozen under-75 °C in a deep-freezer overnight and then samples vials were moved to the freeze-drier (Lablanco-Benchtop, Labconco, USA) and were lyophilized for 12 h. In order to optimize type and concentration of cryoprotectant, lyophilized samples were characterized for ease of redispersibility and thesize, PDI, zeta potential obtained after redispersion.
Various cryoprotectants were screened for optimization of freeze drying process of nanocapsules. Initially, dextrose, sucrose, trehalose and mannitol were added at concentrations of 5% w/v to 2 ml of nanocapsule suspension. After freeze drying the obtained cake was redispersed in 2 mldistilled water and particle size, PDI and zeta potential were analyzed using Zetasizer (Table S1). Size ratio before and after freeze drying with different cryoprotectants was calculated and freeze dried samples were also analyzed for ease of redispersibility. Experimentally it was found that regardless of the nature of the cryoprotectant used, redispersion of the freeze-dried nanocapsules was obtained with all the cryoprotectants in both formulations (CNc, MnosCNc) while, aggregation was observed withoutcryoprotectants. The different means used for redispersion of freeze dried product were manual shaking and vortexing. Vortexing is not acceptable redispersion way for freeze-dried samples. Simple manual shaking (upside downside for 20 s) was found to be sufficient for complete redispersion of the freeze-dried nanocapsules with cryoprotectants. While vortexing was required for 2 min to redisperse freeze dried nanocapsules without cryoprotectant.
Principally, based on minimum size ratio sucrose was found to be most appropriate cryoprotectant (Table S1) and further screening was done to optimize minimum concentration of sucrose sufficient to achieve efficient freeze drying. Table S2 shows the effect of different concentration of sucrose on particle size, PDI and zeta potential. It can be very easily concluded that 2.5 % sucrose provided maximum stabilization to nanocapsule formulations by minimum alterations in size, PDI and zeta potential.
Table S1 Screening of different cryoprotectants
Cryoprotectant(5 % w/v) / Before lyophlization / After lyophlization / Ratio (Sf/Si )
Particle size
(nm) / PDI / Zeta potential
(mV) / Particle size
(nm) / PDI / Zeta potential
(mV)
Dextrose / CNc / 163.8±4.93 / 0.136±0.02 / +29.5±1.20 / 172.2±5.02 / 0.157±0.05 / +31.2±2.10 / 1.051
MnosCNc / 198.9±4.91 / 0.130±0.02 / +32.4±0.91 / 214.2±5.02 / 0.151±0.06 / +34.2±1.31 / 1.076
Sucrose / CNc / 162.3±5.23 / 0.130±0.01 / +27.9±1.10 / 164.1±4.10 / 0.137±0.01 / +29.7±1.12 / 1.011
MnosCNc / 198.6±3.97 / 0.117±0.01 / +31.5±0.69 / 199.8±4.01 / 0.121±0.02 / +32.5±1.62 / 1.006
Trehalose / CNc / 165.5±6.50 / 0.139±0.02 / +29.1±0.98 / 190.2±7.10 / 0.167±0.05 / +33.4±1.52 / 1.149
MnosCNc / 199.2±6.04 / 0.135±0.02 / +33.1±1.01 / 220.2±6.37 / 0.162±0.05 / +35.2±1.30 / 1.105
Mannitol / CNc / 163.4±5.03 / 0.133±0.02 / +29.5±1.03 / 169.8±6.23 / 0.147±0.03 / +30.8±0.72 / 1.039
MnosCNc / 198.2±4.72 / 0.121±0.02 / +32.3±1.13 / 210.4±5.02 / 0.143±0.04 / +33.5±1.04 / 1.061
NCs without
cryoprotectant / CNc / 160.7±7.53 / 0.129±0.03 / +28.2±1.28 / 210.1±8.20 / 0.142±0.01 / +33.8±1.30 / 1.307
MnosCNc / 197.8±8.84 / 0.115±0.04 / +31.7±1.03 / 232.3±6.51 / 0.132±0.02 / +35.8±0.96 / 1.174
Sf/Si -ratio of particle size after freeze drying to particle size before freeze drying
Table S2 Effect of sucroseconcentration
Sucrose concentration(% w/v) / Before lyophlization / After lyophlization / Ratio (Sf/Si )
Particle size(nm) / PDI / Zeta potential(mV) / Particle size(nm) / PDI / Zeta potential
(mV)
1 / CNc / 164.2±4.57 / 0.123±0.02 / +28.6±0.79 / 182.5±4.74 / 0.142±0.03 / +30.6±1.09 / 1.111
MnosCNc / 200.7±5.03 / 0.106±0.04 / +32.3±0.86 / 216.7±4.31 / 0.131±0.02 / +33.7±1.23 / 1.079
2.5 / CNc / 163.6±5.10 / 0.127±0.01 / +28.1±0.94 / 164.8±4.92 / 0.129±0.03 / +28.9±1.08 / 1.007
MnosCNc / 198.9±4.01 / 0.114±0.04 / +31.9±0.75 / 199.4±4.06 / 0.118±0.02 / +32.3±0.93 / 1.002
5 / CNc / 162.3±5.23 / 0.130±0.01 / +27.9±1.10 / 164.1±4.10 / 0.137±0.01 / +29.7±1.12 / 1.011
MnosCNc / 198.6±3.97 / 0.117±0.01 / +31.5±0.69 / 199.8±4.01 / 0.121±0.02 / +32.5±1.62 / 1.006
10 / CNc / 161.8±4.85 / 0.137±0.01 / +27.3±1.32 / 164.1±3.56 / 0.169±0.02 / +28.8±1.69 / 1.014
MnosCNc / 198.9±4.27 / 0.121±0.01 / +28.4±1.36 / 209.3±4.71 / 0.152±0.05 / +30.8±2.06 / 1.052
Aggregation state of amphotericin B (AB)
The toxicity of AB molecule depends on its aggregation state. Thestate of AB within the formulation was assessed by correlating spectroscopic properties with the aggregation state of AB using UV-visible spectroscopy (UV-1700 pharmaSpec, shimadzu). Samples containing 5 µg/ml of AB, were prepared.To assess the state of AB,MnosCNc-AB, CNC-AB and free AB were dispersed in PBS (7.4) and scanned using UV-visible spectrophotometer. AB shows the standard peaks typically at 346, 362, 384 and 408 nm and ratio of the first (346 nm) to the fourth (408 nm) peaks, I/IV (A346/A408), is indicative of the aggregated/monomeric state ratio(aggregation index) which enables analysis of molecular state, directly correlated with toxicity. In case of free AB, I/IV (A346/A408) peak ratio was 1.5,which indicates the presence of aggregated forms of AB. Whereas, A346/A408 ratios for formulated MnosCNc-AB and CNc-ABwere 0.35 and 0.24, respectively,indicates the domination of monomeric state of AB. Nanocapsule architecture prevents self-association of monomeric AB molecules due to packed interacalation of amphiphilic AB molecules among nanoemulsion intermediate generated core and polymer shell, thus reducingthe probability of it undergoing molecular organization to form aggregated states.