Design and Optimization of Doxorubicin Niosomes Derived From

Proniosomes

Srikanth1*, Anand Kumar Y2, Mallikarjuna Setty C3

1*
Research Scholar, JNTU Hyderabad and V.L.College of Pharmacy, Raichur, India.
2
Department of Pharmaceutics, V.L.College of Pharmacy, Raichur, India.
3
Department of Pharmaceutics, Oxford College of Pharmacy, Bengaluru, India.

*Corresponding Author
Srikanth
Assistant Professor
Department of Pharmaceutics
V.L.College of Pharmacy, Raichur.
Karnataka-INDIA-584101
Mobile number: +919160348410
Fax number: +918532240405
E-mail address: srikanthyerigeri@gmail.com
ABSTRACT:
The aim of this research was to design and optimize the doxorubicin niosomes derived from proniosomes using
central composite design. Two independent variables viz., molar ratio of drug to cholesterol (X1), surfactant
loading (X2) and two dependent variables viz., the percentage drug entrapment (PDE) and mean volume
diameter (MVD) were selected for the study. Proniosome were prepared by conventional slurry method and
evaluated for the percentage drug entrapment (PDE) and mean volume diameter (MVD). The PDE dependent
variables and the transformed values of independent variables were subjected to multiple regressions to establish
a second order polynomial equation. Contour plots were constructed to further elucidate the relationship
between the independent and dependent variables. Checkpoint batches were also prepared to prove the validity
of the evolved mathematical model and contour plots. From the computer optimization process and contour
plots, predicted levels of independent variables X1, X2 (0.137, -0.495 respectively), for an optimum response of
PDE (61.427 %) with constraints on MVD (5.712 µm) were determined. The polynomial equations and contour
plots developed using central composite design allowed us to prepare niosomes derived from proniosomes with
optimum responses. After testing different formulations, tween 20 were found to be the best surfactants to form
niosomes.
KEYWORDS: Doxorubicin, Proniosomes, Tween 20, Central composite design, Optimization.
INTRODUCTION:
Development of resistance to chemotherapy, where in drugs elicit suboptimal response to their previously
effective doses in cancer, is one of the major challenges to successful chemotherapy (Choi, 2005; Gottesman,
2002; van Den Elsen et al., 1999). A major mechanism by which cells reduce the intracellular effectiveness of
cytotoxic drugs is by overexpressing either P-glycoprotein (P-gp) or glucosylceramide synthase (GCS) (Liu et
al., 2010; Lucci et al., 1999; Pérez-Sayáns et al., 2010; Ye et al., 2008). P-gp is an efflux transporter whereas
GCS is an enzyme that modifies the efficacy of the internalized drug by modulating intracellular levels of
ceramide.
Choi, C.H., 2005. ABC transporters as multidrug resistance mechanisms and the development of
chemosensitizers for their reversal. Cancer Cell Int. 5.
Gottesman, M.M., 2002. Mechanisms of cancer drug resistance. Annu. Rev. Med. 53,615–627.
van Den Elsen, J.M., Kuntz, D.A., Hoedemaeker, F.J., Rose, D.R., 1999. Antibody C219 recognizes an alpha-
helical epitope on P-glycoprotein. Proc. Natl. Acad. Sci.U.S.A. 96, 13679–13684.
Liu, Y.Y., Gupta, V., Patwardhan, G.A., Bhinge, K., Zhao, Y., Bao, J., Mehendale, H., Cabot, M.C., Li, Y.T.,
Jazwinski, S.M., 2010. Glucosylceramide synthase upregulates MDR1 expression in the regulation of cancer
drug resistance through cSrc and beta-catenin signaling. Mol. Cancer 9, 145.
Lucci, A., Han, T.Y., Liu, Y.Y., Giuliano, A.E., Cabot, M.C., 1999. Multidrug resistance modulators and
doxorubicin synergize to elevate ceramide levels and elicit apoptosis in drug-resistant cancer cells. Cancer 86,
300–311.
Pérez-Sayáns, M., Somoza-Martín, J.M., Barros-Angueira, F., Diz, P.G., Rey, J.M.G., García-García, A., 2010.
Multidrug resistance in oral squamous cell carcinoma: the role of vacuolar ATPases. Cancer Lett. 295, 135–143.
Ye, S., MacEachran, D.P., Hamilton, J.W., O’Toole, G.A., Stanton, B.A., 2008. Chemotoxicityof doxorubicin
and surface expression of P-glycoprotein (MDR1) is regulated by the Pseudomonas aeruginosa toxin Cif. Am. J.
Physiol. Cell Physiol. 295, C807–C818.

Our previous studies with solid lipid nanoparticles (SLN) loaded with MBO-asGCS also demonstrated that
MBO loaded nanoparticles sensitized the resistant NCI/ADR-RES cells to free doxorubicin in the media
(Siddiqui et al., 2010).
Siddiqui, A., Patwardhan, G.A., Liu, Y.Y., Nazzal, S., 2010. Mixed backbone antisense glucosylceramide
synthase oligonucleotide (MBO-asGCS) loaded solid lipid nanoparticles in vitro characterization and reversal of
multidrug resistance in NCI/ADR-RES cells. Int. J. Pharm. 400, 251–259.
Majority of the active pharmaceutical agents currently available in the market and those under development
have poor and variable bioavailability. This problem can be overcome by entrapping the drug into niosomes.
Niosomes are non-ionic surfactant vesicles that can entrap a solute in a manner analogous to liposomes. They
are osmotically active, and are stable on their own, while also increasing the stability of the entrapped drugs 1, 2.
Handling and storage of surfactants require no special conditions. Niosomes possess an infrastructure consisting
of hydrophilic and hydrophobic moieties together and as a result, can accommodate drug molecules with a wide
range of solubilities3. Although niosomes as drug carriers have shown advantages such as being cheap and
chemically stable, they are associated with problems related to physical stability such as fusion, aggregation,
sedimentation and leakage on storage. All methods traditionally used for preparation of niosomes are time
consuming and many involve specialized equipments. Most of these methods allow only for a predetermined lot
size so material is often wasted if smaller quantities are required for particular dose application 4. The
proniosome approach minimizes these problems as it is a dry and free flowing product which is more stable
during sterilization and storage. Ease of transfer, distribution, measuring and storage make it a versatile delivery
system. Proniosomes are water-soluble carrier particles coated with surfactant, which can be measured out as
needed and hydrated to form niosomes immediately before use on brief agitation in hot aqueous media 5-6.
Doxorubicin is antineoplastic drug which is widely used in the treatment of leukemia and various solid tumors,
such as breast, lung and pancreatic cancers. One of the limitations of its use is the non-selective cardiac toxicity
and myelosuppression if administered by injection (11), (1).
1. Jeanneret LJ. The targeted delivery of cancer drugs across the blood-brain barrier: chemical modifications of
drugs or drug-nanoparticles. Drug Discovery Today 2008; 13: 1099-1105.
11. Stan AC, Casares S, Radu D, Walter GF, Brumeanu TD. Doxorubicin-induced cell death in highly invasive
human gliomas. Anticancer Res. 1999; 19: 941-950.
Colorectal cancer is the third most common cancer in the world, with nearly 1.4 million new cases diagnosed in
2012 [1]. Treatments used for colorectal cancer include some combination of surgery, radiation therapy and
chemotherapy [2,3]. Surgery is the primary treatment for patients affected with potentially curable followed by
adjuvant therapy, often suitable in the initial stages; the majority of patients undergo recurrences and metastases.
This phenomenon frequently correlates with an acquired resistance to conventional therapies such as chemo and
radiotherapy [2,3] In metastatic cancer, chemotherapy represents the first-line treatment with the goal of
prolonging survival and improving or maintaining quality of life. Chemotherapeutic drugs, such as doxorubicin,
fluorouracil, cisplatin, leucovorin and mitomycin, are commonly used to kill tumor cells that may have
remained and metastasized or spread to other parts of the body after surgery, but all such drugs have side-
effects, some of them quite serious [2].
Doxorubicin, an anthracycline antibiotic (Fig. 1), has been used for decades for treatment of various types of
cancers [4–8]. While providing a cure in a good degree of cases, doxorubicin is toxic to most major organs,
especially the heart, which renders the treatment dose-limiting [6]. For this reason, many researchers have
designed and developed strategies capable of restricting the toxicity of doxorubicin, to aim its effects directly at
the tumor as much as possible. Promising drug delivery systems include the entrapment of drugs into polymeric
drug carriers, such as hydrogels, nanoparticles, and liposomes [7].
[1] Torre LA, Bray F, Siegel RL, et al. Global cancer statistics, 2012. CA Cancer J Clin 2015;65:87–108.
[2] Labianca R, Beretta GD, Kildani B, et al. Colon cancer. Crit Rev Oncol Hematol 2010;74:106–133.
[3] Manchun S, Dass CR, Cheewatanakornkool K, et al. Enhanced anti-tumor effect of pH-responsive dextrin
nanogels delivering doxorubicin on colorectal cancer. Carbohydr Polym 2015;126:222–230.
[4] Minotti G, Menna P, Salvatorelli E, et al. Anthracyclines: molecular advances and pharmacologic
developments in antitumor activity and cardiotoxicity. Pharmacol Rev 2004;56:185–229.
[5] Menna P, Salvatorelli E, Minotti G. Doxorubicin degradation in cardiomyocytes. J Pharmacol Exp Ther
2007;322:408– 429.
[6] Lyu YL, Liu LF. Doxorubicin cardiotoxicity revisited: ROS versus Top2. In: Liu XY, Pestka S, Shi YF,
editors. Recent advances in cancer research and therapy. Oxford: Elsevier;
2012. p. 351–369.
[7] Tacar O, Sriamornsak P, Dass CR. Doxorubicin: an update on anticancer molecular action, toxicity and
novel drug delivery systems. J Pharm Pharmacol 2013;65:
157–170.
[8] Gutiérrez-Salmeán G, Ceballos G, Meaney E. Anthracyclines and cardiotoxicity. Int J Cancer Res Prev
2015;8:515–521.

Doxorubicin binds to nucleic acids, presumably by specific intercalation of the planar anthracycline nucleus
with the DNA double helix. The anthracycline ring is lipophilic, but the saturated end of the ring system
contains abundant hydroxyl groups adjacent to the amino sugar, producing a hydrophilic center. The molecule is
amphoteric, containing acidic functions in the ring phenolic groups and a basic function in the sugar amino
group. It binds to cell membranes as well as plasma proteins.
It is supplied in the hydrochloride form as a sterile red-orange lyophilized powder containing lactose and as a
sterile parenteral, isotonic solution with sodium chloride for intravenous use only.
Capecitabine is an prodrug of 5'-deoxy-5-fluorouridine (5'-DFUR) figure 1, which is enzymatically converted to
5-fluorouracil in the tumor, where it inhibits DNA synthesis and slows growth of tumor tissue 7,8. It is an orally
administered chemotherapeutic agent used in the treatment of metastatic breast and colorectal cancers9.
Doxorubicin is a cytotoxic anthracycline antibiotic isolated from cultures of Streptomyces peucetius var.
caesius. Doxorubicin consists of a naphthacenequinone nucleus linked through a glycosidic bond at ring atom 7
to an amino sugar, daunosamine. Chemically, doxorubicin hydrochloride is (8S,10S)-10-[(3-Amino-2,3,6-
trideoxy-a-L-lyxo-hexopyranosyl)-oxy]-8-glycoloyl-7,8,9,10-tetrahydro6,8,11-trihydroxy-1-methoxy-5,12
naphthacenedione hydrochloride. The structural formula is as follows: M.W.=579.99.

Figure 1: Structure of doxorubicin

In this research work a simple conventional slurry method was used for the preparation of doxorubicin
proniosomes and optimized. The proniosomes are thus needed to be optimized for the desired response, many
statistical experimental designs have been recognized as useful techniques to optimize the formulation and
process variables10. Different types of experimental design include 3-level factorial design11. D-optimal design12
and Central composite design13. Central composite design requires fewer runs in a 2 factor experimental design
and hence was selected for the present research work. The independent variables selected for the present study
are molar ratio of drug to lipid (X1), surfactant loading (X2). The dependent variables included are PDE and
MVD of niosomes derived from niosomes. The optimization is done using Design Expert 11 (Trial Version 11,
Stat-Ease Inc., Minneapolis, MN) to interpret the results and easy scale up.
MATERIALS AND METHODS:
Materials:
Doxorubicin gift sample was obtained from Shilpa antibiotic Pvt Ltd, Raichur. Maltodextrin was procured from
Himedia, Hosur, cholesterol, tween 20 and DCP (Dicetyl phosphate) were purchased from Loba chem Pvt Ltd,
Mumbai. All the other ingredients and reagents used were of analytical grade.
Method:
Central composite experimental design:
Traditionally pharmaceutical formulations are developed by changing one variable at a time, but this approach
does not give an idea about the interactions among the variables and difficult to develop an optimized
formulation. Hence, a central composite experimental design with 2 factors, 3 levels and 20 runs was selected
for the optimization study. This design consists of 8 full factorial design points, 8 axial points, and 4 center
points. Independent variables with their levels and the dependent variables selected are listed in table 1. The
second order polynomial equation generated from this experimental designs as,
Y1=b0+b1X1+b2X2+b3X3+b12X1X2+b13X1X3+b23X2X3+b11X12+b22X22+b33X32 ..............
Where Y1 is the dependent variable while b0 is the intercept; b1 to b33 are the regression coefficients; and X1, X2
are the independent variables levels of which were selected from the preliminary experiments.
Preparation of proniosomes:
The proniosomes were prepared by the slurry method 14. 250µmol stock solution of surfactant (tween 20) and
cholesterol was prepared in chloroform: methanol (2:1). The accurately measured volumes of tween 20 and
cholesterol stock solutions and doxorubicin (50mg) dissolved in chloroform: methanol (2:1) solutions were
added into a 250ml round bottom flask containing previously 2g of maltodextrin powder used as carrier.
Additional chloroform: methanol (2:1) solution added to form slurry. Further the flask was attached to a rotary
flash evaporator rotated at 60 to 70 rpm. The solvent is allow to evaporate at temperature of 45±2ºC in a reduced
pressure of 600mm/Hg until the mass in the flask had become a dry, free flowing product. The obtained
proniosome powder was further dried overnight in desiccators under vacuum at room temperature. The obtained
dry proniosome powders were stored in air tight amber coloured vials kept in a refrigerator for further
evaluation.
Table 1: Variables and their levels in central composite design.

Independent variables Coded

Low Medium High
X1= Molar ratio of drug lipid -1 0 1
X2= Surfactant loading (Tween 20) -1 0 1

Dependentent variables Goals

Y1= Percentage drug entrapment (%) Maximize
Y2= Mean volume diameter(µm) Minimize
*1.5X corresponds to 15mmol per gram of carrier
These proniosomes were used for preparation of niosomes and characterization of the surface characteristics by
scanning electron microscopy. Proniosomes were transformed to niosomes by hydrating with phosphate buffer
saline (PBS) with a pH of 7.4 at 80°C using vortex mixer for 2 min. The niosomes were sonicated twice for
specified time using a 250-W probe-type sonicator (MAGNA-PAK-250, Libra Ultrasonic, India). Niosomes
were prepared in such a manner that total surfactant concentration remained at 10 mmol in all the batches.
Niosomes were characterized for morphology, PDE and vesicle size in terms of MVD.
Micromeritics properties of proniosomes' powders
The flow properties of doxorubicin proniosomes are vital in handling and processing operations. The flow
properties were studied through measuring the Angle of repose, Carr’s compressibility index and Hausner’s
ratio. The angle of repose was determined by using conventional fixed funnel method. The Carr’s
compressibility index and Hausner’s ratio were calculated from the bulk and tapped density of the proniosomes
powders [1].
1. Cheriyan P, George BJ, Thomas N, Raj P, Samuel J, Carla SrB. Formulation and characterization of maltodextrin based
proniosomes of cephalosporins. World J Pharm Sci 2015;3:62-74.
Db = Wt/bulk volume = W/Vb
Dt = Wt/tap volume = W/Vt
Hausner ratio = D t/D b
Compressibility % = (D t–D b/D t) × 100
Angle of repose Tan Ө = h/r
Rresults
Micromeritics properties of proniosomes' powders
Our results indicated the small angle of repose of prepared doxorubicin proniosomes ranged from 12.8 ° for
FDT13 to 21 ° for FDT7 assuring excellent flow properties. In addition to the angle of repose, Carr’s index
showed a maximum value of 17.8% and the minimum one of 8.4% and Hausner’s ratio were also less than 1.28
ensuring an acceptable flow for proniosomes powder formulations [13].
13. Badawi A, El Nabarawi MA, Elrehem RT, Fayed BA. Formulation and evaluation of dispersed permethrinproniosomes inpowder
and microemulsion-based hydrogel bases for the treatment of scabies. Int J Pharm Pharm Sci 2016;8:221-229.
Scanning electron microscopy:
Proniosomes were sprinkled on to the double sided tape that was affixed on aluminum stubs. The aluminum stub
was placed in the vacuum chamber of a scanning electron microscope (XL 30 ESEM with EDAX, Philips,
Netherlands). The samples were observed for morphological characterization using a gaseous secondary
electron detector (working pressure: 0.8 torr, acceleration voltage: 30.00 KV) XL 30, (Philips, Netherlands).
Table 2: Central composite experimental design with measured responses of doxorubicin proniosomes.
X1 X2 Y1 Y2
Run
A: Cholesterol (%) B: Surfactant (%) PDE (%) MVD(μm)
1 -1 1 58.21±0.25 7.52±0.14
2 1 1 66.21±0.12 5.65±0.52
3 1 -1 67.21±0.22 3.25±0.62
4 1.41 0 71.21±0.33 6.75±0.14
5 0 0 58.21±0.25 6.80±0.17
6 0 0 59.21±0.41 6.85±0.32
7 1 1 68.21±0.17 7.75±0.52
8 -1 1 59.23±0.24 7.85±0.54
9 -1.41 0 55.32±0.32 6.90±0.62
10 0 0 60.21±0.26 6.98±0.14
11 0 -1.41 61.21±0.14 3.25±0.52
12 -1.41 0 56.5±0.22 6.45±0.41
13 1.41 0 72.75±0.25 6.35±0.25
14 -1 -1 60.21±0.32 3.45±0.40
15 0 0 63.21±0.14 6.35±0.41
16 -1 -1 61.21±0.52 3.65±0.21
17 0 1 64.2±0.25 7.95±0.11
18 0 0 65.01±0.52 3.45±0.22
19 1 -1 70.51±0.15 3.95±0.41
20 0 0 66.28±0.32 7.80±0.32
Optical microscopy:
The hydrated niosome dispersions derived from proniosomes were observed using optical microscopy. After
suitable dilution, the noisome dispersions on glass slide and viewed by a microscope (Medilux-207R (II),
Kyowa-Getner, India) with a magnification of 1200X.
Percentage drug entrapment:
The entrapped doxorubicin within niosomes was determined after removing the unentrapped drug by dialysis 15.
The dialysis was carried out by taking niosome dispersion in dialysis tube (donor compartment), which was
dipped in a beaker containing 400 ml of PBS with a pH 7.4 (receptor compartment). The beaker was placed on a
magnetic stirrer run for 4 h with a speed of 80-120 rpm. Then, the solution inside the receptor compartment was
studied for unentrapped doxorubicin at 254 nm using an UV spectrophotometer (UV 1601, Shimadzu, Japan).
The PDE in the niosomes was calculated from the ratio of the difference of the total amount of drug added and
the amount of unentrapped drug detected, to the total amount of drug added.
Zeta potential determination
Particle sizing systems were used in the determination of zeta potential of all formulations. The formulations
were hydrated with distilled water and then converted to niosomes; the formed niosomes were used to determine
the zeta potential by using Particle Sizing System, Inc. Santa Barbara [10].
10. Thomas L, Viswanad V. Formulation and optimization of the clotrimazole-loaded proniosomal gel using 32 factorial design.
Sci Pharm 2012;80:731-48.
Results
Or
Determination of particle size by Zeta sizer (PS):
The PS of the prepared formulations were determined by laser scattering technique using Malvern zeta sizer
(Zetasizer Ver. 7.11, Malvern Instruments UK) after appropriate dilution with double distilled water. The
samples were adjusted to 25°C then subjected to an incident laser beam of 633 nm at a scattering angle of 90°
[21].
21. Abdellatif AA, Tawfeek HM (2015) Transfersomal Nanoparticles for enhanced transdermal delivery of
clindamycin. AAPS Pharm SciTech: 1-8.

Results:
Particle-size analysis and zeta potential determination:
Particle-size analysis of the proniosome-derived niosomes shows that the average ± SD(nm) of particle size of
90% of the particle is 60_170 nm with polydispersity index of 0.3167.
 Figure: Particle size determination optimized batch

Figure: Zeta potential determination optimized batch

Measurement of vesicle size:

The vesicle dispersion was diluted about 100 times in the same buffer used for their preparation. Vesicle size
was measured on a particle size analyzer (Laser diffraction particle size analyzer, Sympatec, Germany). The
apparatus consists of a He-Ne laser beam of 632.8 nm focused with a minimum power of 5 mW using a Fourier
lens [R-5] to a point at the center of multielement detector and a small volume sample holding cell (Su cell). The
sample was stirred using a stirrer before determining the vesicle size.
RESULTS AND DISCUSSION:
Morphology of dry proniosomes and niosomes derived proniosomes:
Proniosomes were prepared by the slurry method using maltodextrin as a carrier. Scanning electron microscopy
(SEM) of uncoated maltodextrin powder (figure 2a) shows the highly porous surface, which would provide
more surface area to be coated with surfactant mixture. Proniosomes were made with different proportions of
drug and surfactant coating (figure 2b, c and d) is SEM images of different proniosome batches made at
different surfactant loading. Surface of the proniosomes batches FDT1 and FDT20, made at 1.5X and 3X
respectively, was observed as being smooth and uniform while that of batch PDT14, made at 4.5X surfactant
loading was seen rough, thick and uneven. Morphology of proniosome derived niosomes was studied under
optical microscope. Niosomes prepared from proniosomes were spherical in shape (figure 3)

Figure 2: SEM of proniosomes prepared: (a) with pure maltodextrin, (b) at 1.5x surfactant loading, (c) at 3x surfactant loading, and
(d) at 4.5x surfactant loading.

Figure 3: Optical photomicrograph of niosome-derived from proniosomes (batch fdt14).

Optimization study of proniosomes:
An optimization using central composite design for 2 factors, 3 levels offers an advantage of fewer experimental
runs (20 runs). The experimental runs and the observed responses for the 20 batches are given in table 2. The
different levels of independent variable combinations resulted in different PDE and MVD values. The PDE
values observed were in the range of 56.32% in batch FDT9 (minimum) to 72.75% in batch FDT13 (maximum).
The Model F-value of 22.37 implies the model is significant. There is only a 0.01% chance that an F-value this
large could occur due to noise.P-values less than 0.0500 indicate model terms are significant. In this case A, A²,
B² are significant model terms. Values greater than 0.1000 indicate the model terms are not significant. The
Lack of Fit F-value of 2.22 implies the Lack of Fit is not significant relative to the pure error. There is a
14.26% chance that a Lack of Fit F-value this large could occur due to noise. Non-significant lack of fit is good,
we want the model to fit. This indicates selected two independent variables have a profound effect on the PDE
within niosome derived from proniosomes. The second order polynomial equation relating the response PDE
and the independent variables was:
PDE=60.21+4.9208A-0.0784B+0.085AB+1.821A2+1.936B2.........................
The values of the coefficients X1-X2 are related to the effect of these variables on the PDE. Coefficients of more
than one terms represents interaction and show how the response changes when two factors are simultaneously
changed. Coefficients of higher order terms represent quadratic relationship and are included to investigate
nonlinearity. The polynomial equation can be used to draw conclusions after considering the magnitude of each
coefficient and the mathematical sign it carries (i.e., positive or negative). The high value (0.88) of correlation
coefficient (R2) in above equation indicates a good fit. Proniosomal batches FDT4, FDT13 and FDT19 exhibited
high PDE value, i.e. more than 70% (table 2). A negative sign of coefficient for molar ratio of drug: lipid (X 1)
and surfactant loading (X2) represents antagonistic effect of these variables. In this study at different levels of
X1, lipid was kept constant and the amount of drug was increased for each level to give a different molar ratio.
So at a low level of X1 high PDE value might be due to more availability of lipophilic ambience for the drug
entrapment. The significance of the different formulation variables and their interactions was compared using
analysis of variance (ANOVA) at a significance level of p<0.05. From the P value for PDF analysis given in
table 3, it can be concluded that the molar ratio of drug: lipid have significant effects on the PDE of doxorubicin
proniosome-derived niosomes and no interaction term has a significant effect on the PDE.
Table 3: Summary of results of Analysis of variance for PDE.
Sum of Mean
Source df F-value p-value
Squares Square
Model 432.87 5 86.57 22.37 < 0.0001 significant
A-cholesterol 387.43 1 387.43 100.09 < 0.0001
B-surfactant 0.0985 1 0.0985 0.0254 0.8755
AB 0.0578 1 0.0578 0.0149 0.9045
A² 30.33 1 30.33 7.83 0.0142
B² 34.28 1 34.28 8.86 0.0100
Residual 54.19 14 3.87
Lack of Fit 20.46 3 6.82 2.22 0.1426 not significant
Pure Error 33.73 11 3.07
Cor Total 487.06 19
Vesicle size (MVD) of the niosome batches was measured by low angle laser light scattering technique and was
found to be in the range of 3.25μm to 7.85μm. A polynomial equation was developed for MVD, described as:
MVD=6.745-0.138A+1.704B-0.258AB-0.248A2-0.748B2.......................
The value of the correlation coefficient (R2) in above equation was found to be 0.90 indicating a good fit. A
positive sign of the coefficients for the molar ratio of drug: lipid and surfactant loading indicates favorable
effects on MVD. Positive effects of X1 could be attributed to hydrophobic interaction between drug and
surfactant. Favorable effect of X2 may be due to efficient hydration of the uniform and thin film of surfactant at
low surfactant loading compared to the film obtained at a high surfactant loading.
As shown in table 4, among the independent variables selected the terms X 1 and X2 were found to be significant
(P<0.05) in predicting the MVD. The Model F-value of 27.20 implies the model is significant. There is only a
0.01% chance that an F-value this large could occur due to noise.P-values less than 0.0500 indicate model terms
are significant. In this case B, B² are significant model terms. Values greater than 0.1000 indicate the model
terms are not significant. If there are many insignificant model terms (not counting those required to support
hierarchy), model reduction may improve your model.The Lack of Fit F-value of 3.02 implies there is a 7.55%
chance that a Lack of Fit F-value this large could occur due to noise. Lack of fit is bad -- we want the model to
fit. It is also evident from table 4 that the quadratic effects of all the independent variables i.e. X12, X22 have
significant effects on MVD.
Table 4: Summary of results of Analysis of variance for MVD.
Source Sum of Squares df Mean Square F-value p-value
Model 52.50 5 10.50 27.20 < 0.0001 significant
A-cholesterol 0.3090 1 0.3090 0.8004 0.3861
B-surfactant 46.47 1 46.47 120.38 < 0.0001
AB 0.5356 1 0.5356 1.39 0.2585
A² 0.5643 1 0.5643 1.46 0.2467
B² 5.12 1 5.12 13.27 0.0027
Residual 5.40 14 0.3861
Lack of Fit 2.44 3 0.8142 3.02 0.0755 not significant
Pure Error 2.96 11 0.2693
 Cor Total 57.90 19
As the central composite design includes two center points, we can estimate the pure error of the experiments
and enable the model’s to be checked for lack of fit. For the experimentally obtained data, the test for lack of fit
did not yield statistical significance (P>0.05), and the results indicated that the models for PDE and MVD were
satisfactory (Table 3 and 4).
Three-dimensional and Contour plots:
Presentation of the data as graphs can help to show the relationship between the independent and dependent
variables. Figure 4 is a 3D and contour plot drawn at 0 levels showing the effect of X1 and X2 on MVD and PDE
of proniosome-derived niosomes. The contours for all the values of MVD were found to be nonlinear. It was
evident from figure 4 that low value of MVD could be obtained with low level of both X 1 and X2 and that high
values of PDE (≥72%) can be obtained for different combinations of the two independent variables, X1 in the
range of less than -0.8 level and X2 in the entire range of -1 level to 1 level.

Figure 4: Three-dimensional and contour response surface plot image showing influence of independent variables on PDE and
MVD.
After studying the effects of the independent variables on the responses, the levels of these variables that give
the optimum responses were determined. The optimum formulation is one that gives a high value of PDE
(≥70%) and is constrained to a low MVD (≤ 5 μm) as well as having a high total amount of drug entrapped and
low amount of carrier present in the resultant niosomes. Using a computer optimization process and the contour
plots shown in Figure 4, the levels selected for both X1 and X2 were 0.137 and -0.495 respectively, which gives
the theoretical value of 61.427% and 5.712μm for PDE and MVD, respectively. Decreasing the level of X2 from
the optimum level resulted in a significant increase in the amount of carrier but an insignificant increase in the
PDE value. However, an increase in the level of X1 above the selected level led to an increase in the PDE value
but as well an increase in the vesicle size above the desired value. Hence, 0.137 level of molar ratio of drug:
lipid (X1), -0.495 level of surfactant loading (X2) was selected as optimum. For a confirmation, a fresh
formulation was prepared at the optimum levels of the independent variables and the resultant proniosomes were
transformed to niosomes and evaluated for the responses. The observed values of PDE and MVD were found to
be 61.21% (FDT11) and 5.62μm (FDT2) respectively, which were in close agreement with the theoretical
values.
Checkpoint analysis:
Three checkpoint batches were prepared and evaluated for PDE and MVD as shown in table 5. Results indicate
that the measured PDE and MVD values were as expected. When obtained responses were compared with the
predicted PDE and MVD value from coefficients and contour plots, using student’s t-test, the differences were
found to be insignificant (P> 0.05). Thus, we can conclude that the obtained coefficients and plotted contour
plots are valid in predicting the levels of independent variables for desired response.
Table 5: Checkpoint Batches of doxorubicin proniosomes with their Measured and Predicted value of
PDE and MVD.
Y1 Y2
Batches X1 X2
Measured* Predicted Measured* Predicted
1 0.137 -0.495 61.42 58.88 5.71 4.16
2 1.000 1.000 68.89 68.89 7.05 7.05
3 -1.000 1.000 58.88 68.88 7.85 7.85
CONCLUSION:
Slurry method was found to be simple and suitable for laboratory scale preparation of doxorubicin proniosomes.
Optimization of a proniosome formulation is a complex process that requires one to consider a large number of
variables and their interactions with each other. The present study conclusively demonstrated the use of a central
composite design in optimization of proniosome batches. After testing different formulations, tween 20 were
found to be the best surfactants to form niosomes. The derived polynomial equations and contour plots aided in
predicting the values of selected independent variables for the preparation of optimum proniosome batches with
desired properties.
ACKNOWLEDGEMENT:
The authors are thankful to Shilpa antibiotics, Raichur, India for providing gift sample of doxorubicin and
Principal, Management, teaching and non teaching staff of VL College of Pharmacy, Raichur for encouragement
and support in carrying out the work.
CONFLICT OF INTEREST
Authors declared none.
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