Hyaluronidase is an enzyme that catalyzes breakdown of hyaluronic acid. This property is utilized for hypodermoclysis and for treating extravasation injury. Hyaluronidase is further studied for possible application as an adjuvant for increasing the efficacy of other drugs. Development of suitable carrier system for hyaluronidase would help in coadministration of other drugs. In the present study, the hyaluronidase was encapsulated in liposomes. The effect of variables, namely, phosphatidylcholine (PC), cholesterol, temperature during film formation (
Hyaluronic acid (HA) is a polysaccharide containing alternating units of glucuronic acid and glucosamine [
Use of hyaluronidase as an adjuvant therapy for improving the pharmacokinetic properties of coadministered drug is of particular interest as many of the regulatory bodies including US FDA have approved its use in humans [
Understanding the effect of variables on the outcome is important for developing a product with all the predetermined requirements [
Liposomal delivery of proteins is one of the widely explored methods for delivery of proteins. In the present study, hyaluronidase was encapsulated in liposomes. The effect of process variables on size, percentage drug encapsulation, and zeta potential was studied using factorial design and CCD.
Cholesterol (extra pure) and chloroform (HPLC grade) were purchased from SRL Pvt. Ltd., (India) and Merck (India), respectively. L-α-phosphatidylcholine and triton-X 100 were obtained from Sigma (India). Lyophilized hyaluronidase (Hynidase Injection I.P. (Ovine), marketed by Shreya Life Sciences Pvt. Ltd., India) was used as drug source.
Hyaluronidase loaded liposomes were prepared using thin film hydration method. Lipids (Table
Fractional factorial design showing the effect of independent variables on the outcome.
Run order | PC (A) (%w/v) | Ch (B) (%w/v) |
|
SPR (D) rpm | % PE |
---|---|---|---|---|---|
1 | 50 | 0.5 | 40 | 200 | 10.22 |
2 | 200 | 0.125 | 40 | 200 | 7.69 |
3 | 200 | 0.5 | 40 | 50 | 5.51 |
4 | 50 | 0.125 | 60 | 200 | 9.7 |
5 | 200 | 0.125 | 60 | 50 | 8.38 |
6 | 50 | 0.5 | 60 | 50 | 10.38 |
7 | 200 | 0.5 | 60 | 200 | 6.17 |
8 | 50 | 0.125 | 40 | 50 | 10.58 |
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Effect | −3.28 | −1.02 | 0.16 | −0.27 | NA |
AB—1.18 | AC—0.52 | AD—0.25 | NA | ||
% cont. | 79.12 | 7.59 | 0.18 | 0.52 | |
AB—10.18 | AC—1.96 | AD—0.46 | NA | ||
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OWA | 0.003 | 0.51 | 0.921 | 0.87 | |
TWA | AB—0.022 | AC—0.55 | AD—0.78 | NA |
PC: phosphatidylcholine; Ch: cholesterol;
OWA: one-way ANOVA; TWA: two-way ANOVA; AB, AC, and AD: interaction between phosphatidylcholine-cholesterol, phosphatidylcholine-temperature, and phosphatidylcholine-speed of rotation, respectively. NA: not applicable.
Liposomes formed after overnight hydration were sonicated at 60% amplitude control (probe sonicator, Sonics and materials Inc., USA) for total duration of 30 sec consisting of three cycles. Duration of each cycle was 10 sec with 10 sec interval. The formulation was maintained on ice bath during sonication. The liposomes were then transferred into polypropylene tubes and centrifuged at 450 g for 3 min to remove the coarse particles. The supernatant was then centrifuged at 34,600 g for 60 min (Sigma Laborzentrifugen 3k30, Germany) to collect the nanoparticles. The pellet obtained was used for calculating the particle size and percentage drug encapsulation. For size measurement pellet was dispersed in ultrapure water (Milli-Q, Millipore) and analyzed using photon correlation spectroscopy (Malvern Zetasizer). Nanoliposomes were made to release entrapped protein by treating them with 1% triton X-100 [
The effect of variables involved in preparation of hyaluronidase loaded nanoliposomes was studied in two stages. First the effect of PC, cholesterol, temperature during film formation (solvent evaporation) (
Factorial studies showed that the outcome, that is, percentage of protein encapsulated, was significantly affected by the amount of PC present in the system. One-way ANOVA showed that the effect of PC on percentage of drug encapsulation was highly significant with a
The outcome had very high dependence on the level of PC to an extent that the outcome was not affected by the level of other independent variables, namely,
Contour plot for factorial studies. (a) The effect of various levels of phosphatidylcholine and cholesterol on percentage protein encapsulation when the level of the other variables was maintained at their lowest level. (b) The effect of various levels of phosphatidylcholine and cholesterol on percentage protein encapsulation when the level of the other variables was maintained at their highest level. (c) The effect of various levels of temperature (during film formation) and speed of rotation of the flask (rpm) on percentage protein encapsulation when the level of the other variables was maintained at their lowest level. (d) The effect of various levels of temperature (during film formation) and speed of rotation of the flask (rpm) on percentage protein encapsulation when the level of the other variables was maintained at their highest level.
Box plot showing the effect of two-way interaction of independent variables on the outcome. (a) shows the effect of interaction of cholesterol and phosphatidylcholine on percentage protein encapsulation. (b) shows the effect of interaction of rpm and phosphatidylcholine on percentage protein encapsulation.
CCD study showed that PC had significant effect (
Central composite design showing the effect of independent variables on the dependent variable.
Std. order | Run order | PC (A) (%w/v) (A) | SPR (rpm) (B) | % PE | Size (nm) | ZP (mV) |
---|---|---|---|---|---|---|
3 | 1 | 40 | 225 | 9.58 | 562.9 | −49.2 |
5 | 2 | 22.40 | 137.5 | 10.02 | 1388 | −42.9 |
7 | 3 | 82.5 | 13.76 | 9.00 | 837.8 | −60.1 |
8 | 4 | 82.5 | 261.24 | 9.42 | 505.2 | −54 |
6 | 5 | 142.60 | 137.5 | 8.60 | 273.8 | −59.6 |
9 | 6 | 82.5 | 137.5 | 8.67 | 557.2 | −64.3 |
10 | 7 | 82.5 | 137.5 | 8.93 | 417.8 | −51.3 |
2 | 8 | 125 | 50 | 8.67 | 558.3 | −58.6 |
12 | 9 | 82.5 | 137.5 | 8.49 | 1110 | −65 |
1 | 10 | 40 | 50 | 9.93 | 1978 | −56.4 |
4 | 11 | 125 | 225 | 8.27 | 737 | −64.8 |
11 | 12 | 82.5 | 137.5 | 8.11 | 446.5 | −63.3 |
13 | 13 | 82.5 | 137.5 | 8.96 | 514.3 | −51.3 |
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PC (A) | 0.005 | 0.006 | 0.032 | |||
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SPR (B) | 0.902 | 0.06 | 0.57 | ||
A * B | 0.961 | 0.02 | 0.276 |
PC: phosphatidyl choline; SPR: speed of rotation (during film formation); % PE: percentage of protein encapsulation; ZP: zeta potential.
Although both the independent variables (PC and SPR) could affect the outcome, SPR is a physical parameter whose effect is always going to depend on the level of PC. SPR affects the distribution and homogeneity of the film within the flask [
The percentage of protein encapsulated in all the trials during CCD was between 8 and 10% (Table
Surface plot for CCD studies showing the effect of interaction of phosphatidylcholine and speed of rotation of flask during film formation (rpm) on percentage of protein encapsulation (a) and size (b) when the level of other independent variables was maintained at a predetermined level.
Overlaid contour plot showing the effect of interaction of phosphatidylcholine and speed of rotation of the flask (rpm) during film formation on size, percentage protein encapsulation, and zeta potential.
The study on the effect of independent variables on zeta potential showed that PC had significant influence on the outcome (Table
Size (a) and zeta potential (b) of hyaluronidase loaded nanoliposomes prepared using optimized conditions.
Experimental design was used to identify the main variables with significant effect and effect of interaction among the individual variables on the outcome in terms of drug encapsulation efficiency, mean particle size, and zeta potential of nanoliposomes containing hyaluronidase. Fractional factorial study showed that phosphatidylcholine was the critical component whose level determines the percentage of protein encapsulation. The ideal conditions for production of particles with least possible and maximum encapsulation efficiency and good zeta potential were studied and identified using CCD. Under the optimized conditions (phosphatidylcholine − 140 mg, cholesterol − 1/5th of phosphatidylcholine, temperature during film formation − 50°C, and speed of rotation of flask during film formation − 150 rpm) the mean particle size was found to be
All the authors of this paper declare that there is no conflict of interests with any financial organization regarding the material discussed in this paper.
The authors would like to acknowledge the authorities of Manipal University for providing the required facilities to carry out this experiment.