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IJAPR / July 2014/ Vol. 5 /Issue. 7 / 332– 347 1

R.Sureshkumar, et al. / International Journal of Advances in Pharmaceutical Research

IJAPRAvailable Online through

www.ijapronline.orgResearch Paper

I SSN: 2230 – 7583

PHYSICO-CHEMICAL CHARACTERIZATION AND DEVELOPMENT OF POLYMER COATED IRON NANOPARTICLES

FOR TREATMENT OF ANAEMIAR.Sureshkumar, Y.Anil Raju, S.Sai Ratan

Assistant Professor, Department of Pharmaceutics, JSS College of Pharmacy, Ootacamund, Nilgiris, TamilNadu.643001

Received on 03 – 04 - 2014 Revised on 15 – 05- 2014 Accepted on 11– 06 – 2014

ABSTRACTThe aim of this investigation is to improve the pharmacological activity of iron with the development of

biocompatible nanoparticle for intravenous drug delivery of iron for useful in the treatment of anemia. These nanoparticles are highly reactive because of their large surface area. In the presence of oxygen and water, they rapidly oxidize to form free iron ions. Aggregation of iron particles generally occurs due to its magnetic behavior. To prevent these aggregation of iron particles were coated with polymer. In this investigation we done the preformulation studies for understanding the interaction between the active ingredient with the excipients. In this investigation includes various tests are followed PH, particle size distribution (PSD), Osmolality (mOsm), conductivity, polymer content, extraction/separation of CP, Fourier transform infra-red spectroscopic studies (FTIR) were carried out for the study. For the formulation development includes various parameters are oxidation of carbohydrate polymer- effect of sodium hypo chloride (NaOCl) concentration on the oxidation of CP (starch) from material stock, effect of PH on oxidation of CP (starch) from material stock, effect of time on oxidation of CP (starch) from material stock, effect of oxidation of polymer (starch) on conductivity, analysis of oxidation by FTIR spectra, preparation of starch coated Fe-nanoparticles using polymer (starch) oxidized at variable NaOCl concentration, preparation of starch coated Fe-nanoparticles using different iron to polymer (starch) ratio, correlative development of Fe-NPs by alterations in PH and temperature. The obtained results are indicated Fe-nanoparticles were found in the particle size of 40.21 nm, it was similar to desired particle size range (25nm-45nm) it was comparable with RMP. We carried out stability of Fe-Nanoparticles (Fe-NPs) at different temperature ranges, even upto 900C, it showed no changes in peaks done by FTIR studies.Key Words: Iron Nanoparticles, Particle size distribution, polymer coating,

1. INTRODUCTIONNanoparticles are defined as particulate

dispersions or solid particles with a size in the range of 10-1000nm1.Nanotechnology is the sixth truly revolutionary technology introduced in the modern

Author for Correspondence R.SureshkumarAssistant Professor Department of Pharmaceutics JSS College of Pharmacy Ootacamund, The Nilgiris TamilNadu.643 001. [email protected] Mobile No: +919865064872

world following the industrial revolution of the mid 1700s, and bio-technology revolution of the 1900s.Nanobiotechnology is door to future, nano biotechnology is the unification of biotechnology and nanotechnology2.Nanomaterials have unique physicochemical properties, such as ultra-small size, large surface area to mass ratio, and high reactivity, which are different from bulk materials of the same composition3.Nanotechnology drug therapies are proving to cause fewer side effects and increase effectiveness over traditional therapies. Nanotechnology is expected to make an immeasurable impact on medical diagnosis and


treatment4.



The Nanoparticles are divided into following types according to their physical sizes. Liquid crystals, liposomes, nanocrystals, solid lipid nanoparticles, polymeric nanoparticles, and dendrimers5-6.

Anemia is a blood disorder it occurs when there is not enough hemoglobin in a blood. Iron is the most common cause of anemia. Generally anemia called as iron deficiency anemia. It occurs due to blood loss, lack of RBCs and destruction of RBCs. RBCs produce heamoglobin with help of erythropoietin. Heamoglobin contains iron as major constituent. The main causes for anemia is blood loss, decreased red blood cell production, and destruction of red blood cells7.

The types of anemias are hemolytic anemia, iron-deficiency anemia, and vitamin-deficiency anemia and folic acid deficiency anemia7-8. Anemia also caused by the inherited problems those are sickle cell anemia and thalassemia. Anemia also caused by the other diseases, those are cancer, kidney diseases and immune system diseases, such as rheumatoid arthritis and lupus8.

Some applications regarding the nanoparticles are, nanoparticles can better deliver drugs to tiny areas with in the body, nanoparticles overcome the resistance offered by the physiological barriers in the body because efficient delivery of drug to various parts of the body is directly affected by particle size, and targeted nano drug carriers reduce drug toxicity and provide more efficient drug distribution9. In the last decade, super paramagnetic iron oxide (SPIO) nanoparticle has become the gold standard for MRI cell tracking, and has even entered clinical use10. Nanotechnology has provided an advance biomedical research tool in diagnostic imaging, therapy and targeting of NPs to individual cells and sub cellular compartment11.

These iron nano particles circulates entire body through blood stream without any blocking. Administration of large molecules cause accumulation inside the body during administration. Nanoscale iron particles are sub-micrometer particles of iron metal12-13. They are highly reactive because of their large surface area. In the presence of oxygen and water, they rapidly oxidize to form free iron ions. They are widely used in medical and laboratory applications. Aggregation of iron particles generally occurs due to its magnetic behavior13. To prevent these aggregation of iron particles were coated with polymer. Polymeric nanoparticles are ideal vehicle for many controlled delivery applications due to their ability to encapsulate a variety of drugs. In this instance iron nanoparticles are boon to anemic patient with problem in using of oral iron therapy14-15. The

present work deals with the development of biocompatible nanoparticle for intravenous delivery of iron for the treatment of anemia.

2. MATERIALS AND METHODSFerric chloride hexahydrate, carbohydrate

polymer and sodium bromide were received from Sigma-Aldrich Corporation, sodium hydroxide, sodium hypo chloride and methanol were received from Merck chemicals, sodium carbonate anhydrous was received from S.D fine chem. Ltd, Mumbai, ethanol absolute was received from Changshu- Yangyuan chemical Co., Ltd, hydrochloric acid was received from J.T baker organic reagent chemicals. All the chemicals were of AR grade.

Preformulation studies 16

Preformulation studies were done to assess the interaction between the active ingredients and the excipients. In this research PH, particle size distribution, osmolality, conductivity, polymer content, extraction/separation of carbohydrate polymer (CP), Fourier transform infra-red spectroscopic studies (FTIR) were carried out for the study. Preformulation study in the present investigation includes various tests carried out for characterization of carbohydrate polymer and iron salt in comparison to reference medical product (RMP).

Physico-chemical characterization of reference medical product (RMP), starch (CP), extracted CP from RMP (Ex-CP)

Reference medical product (RMP), starch from material stock (CP), extracted CP from RMP (Ex- CP), were characterized for following parameters to correlate all the parameters for different materials (RMP, CP and Ex-CP) thus it can be used as reference standard for further formulation and process optimization and development.2.1.2 PH 17-18

PH reflects a solution to be acidic or basic, PH is directly proportional to OH- concentration and inversely proportional to H+ ion concentration. The PH of the RMP and starch from material stock (CP) and extracted polymer (Ex-CP) from RMP, were measured by PH meter (Mettler Toledo, Seven Multi). 100mg of each were taken and dissolved in 5ml water for injection (WFI) each in fresh falcon tube then the PH of each sample was measured. The temperature of measurement was done at 25.0 ± 0.5oC.

Particle size distribution (PSD) 19

The study of particle size works on the principle of scattering of light from the surface area of the particle when a beam of laser strikes the particles which are in brownian motion. The change in intensity of scattered light was correlated with that size of the particles present in the solution. 30 µl of



RMP was diluted to 3ml in the measurement cell (Cuvette). The cuvette was loaded into the particle size analyzer (Zetasizer, Nano-S Malvern Instrument). Similarly CP (starch) from material stock (100mg) was weighed and dissolved in 3ml WFI which was poured into cuvette and particle size was measured. The data representation was in the form of size distribution curve showing particle size distribution (nm) against Intensity (%).

Osmolality 20

Osmolality (mOsm) is the measurement of solute concentration in the solution being tested. It was executed to observe the effect of solute concentration. The osmolality of the RMP, CP and Ex-CP were measured using calibrated osmometer (Advanced Instrument Model 3250). The standard solution of 290 mOsm was measured to check instrument precision followed by measurement of osmolality of samples. The results were observed to find out the degree of ionization due to solute concentration.

Conductivity 21-22

The conductivity determines the degree of ionization and holds a correlation with oxidation process and ionization taking place during adjustment of PH with acid and alkali. The conductivity of the RMP (1ml), CP (Starch) from material stock (100mg) and extracted CP (Ex-CP) from RMP (10mg) was measured using conductivity meter (Mettler Toledo, Seven Multi).

Polymer content 23

In order to find out polymer content from the RMP, 1ml of RMP was pipetted into activated dialysis bag. Water for injection (WFI) was used as hypotonic solution for the movement of salts was replaced at regular interval of 30min. Dialysis was continued till the PH of WFI showed a constant reading. Then the PH of dialyzed RMP was measured and then lyophilized. The weight of the dialyzed & lyophilized RMP was measured. Iron content of RMP (50mg/ml) as mentioned on the product leaflet was subtracted from weight of dry powder to calculate the polymer content.

Extraction/separation of CP (RMP) 24-25

Polymer was coated on the iron particles with the help of hydrogen bonding and vander wall force of attraction in RMP. Carbohydrate polymer was extracted from the RMP to characterize it by different methods. 1ml of RMP was taken in 15ml falcon tube and to that 1ml of 30% of HCL was added and incubated at 50oC for 15min followed by cooling to RT. 3times absolute ethanol was added and vortexes followed by centrifugation and decantation of supernatant. The obtained pellet was redispersed in WFI followed by measurement of PH and osmolality and compared with that of CP.

Fourier Transform Infra-Red Spectroscopic Studies (FTIR) 26-27

Infrared spectroscopy is an important technique. It is an easy way to identify the presence of certain functional groups in a molecule. Also, one can use the unique collection of absorption bands to confirm the identity of a pure compound or to detect the presence of specific impurities. Infrared spectral matching approach was employed to detect the possible effect of oxidation of the functional group present on the polymer and thus the spectra profile of CP, Ex-CP and RMP were measured and results were compared. CP, Ex-CP and RMP were freeze dried before mixing with KBr to prevent any disturbances in IR spectra. 10mg sample was weighed and mixed with 1gm KBr in a preheated mortar and pestle. After mixing, sample was hand pressed into the loader then it was scanned in mid IR region from 400-4000cm in FTIR spectrophotometer (Shimadzu 8400S) and IR spectra was measured to identify appearance and disappearance of different peaks upon oxidation.

Characterization of iron salt (ferric chloride hexahydrate, Fecl3.6H2O)

PH

The PH of the solution was taken by using PH meter (Mettler Toledo, Seven Multi) 500mg of the Iron salt was dissolved in 5ml of WFI. The solution was vortexed for 5min and PH was measured. This procedure was performed in triplicates to get the accurate value.

Formulation developmentFormulation development deals with optimization

of various process and formulation parameters and correlation of observations and data obtained from the preformulation studies for the optimization and development of desired formulation. In the present research we are trying to develop Fe hydroxide- nanoparticles coated with oxidized carbohydrate polymer (Starch). There are 3 major steps to get the desired formulation.

1. Oxidation of polymer2. Synthesis of iron nanoparticles3. Coating of polymer on nanoparticles

We have optimized various process andformulation parameters at each step to achieve formulation with desired characteristics with reference to RMP.

Oxidation of carbohydrate polymer 28

Effect of sodium hypo chloride (NaOCl) concentration on the oxidation of CP (starch) from material stock

To oxidize polymer an oxidizing agent is required which can be easily acquired, cost effective, special storage should not be required, and foremost point is it should oxidize the CP (starch) from material stock



to the required and optimum levels to achieve desired



iron nanoparticles (Fe NPs). Sodium hypochlorite (NaOCl) is an oxidizing agent which cause the conversion of primary alcohols into aldehyde it is due to presence of water, aldehyde group is converted into carboxylic group. This is the basic oxidation process and NaOCl was used as oxidize polymer.

Various quantities form 0 µl - 1000µl of NaOCl was used to oxidize the carbohydrate polymer. The polymer concentration was 1gm/ml. The result was screened by comparing the end PH with the obtained PH of the polymer extracted (Ex-CP) from the RMP.

Effect of PH on oxidation of CP (starch) from material stock29

The PH was adjusted to acidic (4.0) with 30% HCl, neutral (7.0) and basic (9.5) with NaOH, after addition of NaOCl (270µl) into the solution containing CP (starch). Three solutions were kept at RT and the PH of the solutions was measured at regular intervals. After 4 hrs the polymer was purified by ethanol purification and redispersed into WFI. PH was again measured after redispersion for each solution. The results were correlated with PH of Ex-CP and utilized for further optimization.

Effect of time on oxidation of CP (starch) from material stock30

Time required for the oxidation of polymer is a very critical parameter to achieve desired oxidation with respect to RMP an Ex-CP. The effect of time on oxidation was studied by oxidizing solution of 1gm of CP (starch) with variable volumes of NaOCl (0- 1000µl). The effect of time on the oxidation was observed by measuring the PH at regular intervals of time.

Effect of oxidation of polymer (starch) on conductivity31

In order to study the effect of oxidation on conductivity different samples of CP (starch) (100mg/5ml) was subjected to oxidation with varying volume of NaOCl (50µl - 5000µl) for 4 hrs. After 4 hrs, conductivity was measured for each sample which showed the amount of ions present and thus extent of oxidation. The obtained result were compared with the conductivity of Ex-CP.

Analysis of oxidation by FTIR spectraInfrared spectroscopy is an important technique. It

is an easy way to identify the presence of certain functional groups in a molecule. Also, one can use the unique collection of absorption bands to confirm the identity of a pure compound or to detect the presence of specific impurities Infrared spectral matching approach was employed to detect the possible effect of oxidation on the functional group present and it can be compared with that of unoxidised carbohydrate polymer (polymer (starch) for material stock) and also with RMP it can indicate qualitative effect of oxidation on the polymer.

CP extracted from RMP was freeze dried before mixing with KBr to prevent any disturbances in IR spectra. 10mg sample was accurately weighed and it was mixed with 1gm of KBr in a preheated mortar and pestle. After mixing sample was transfer to hand pressed loader then it was scanned in mid IR region from 400-4000/cm in FTIR spectrophotometer (Shimadzu 8400S) to get IR spectra and to check the appearance and disappearance of peak upon oxidation, similar procedure was followed in CP (starch) was oxidized for 240min (4hrs) using NaOCl volumes are 270µl and 500µl.

Preparation of starch coated Fe-nanoparticles using polymer (starch) oxidized at variable NaOCl concentration

100gm of Fe-salts were weighed (20% Fe) and it was dissolved in 120ml of water, where CP (starch) was weighed 4gm for each volume of NaOCl and it was dissolved in 10ml of water, different volume of NaOCl were used (200,250,270,300,500)µl according to result obtained .CP (starch) sample was withdrawn (2.5ml) after 1hour, similarly CP (starch) (2.5ml) was withdrawn after 2, 3 and 4 hours for all the volume of NaOCl used. Thus obtained results were used for further optimization of parameters for production of Fe-nanoparticles.

Preparation of starch coated Fe-nanoparticles using different iron to polymer (starch) ratio

The different ratios (1:1.5, 1:2 and 1:3) of iron to polymer were used for the development of iron nanoparticles. Each molecule of iron salt consists of 20% of iron. The iron to polymer amount was weighed. Three batches were made. Fe-salt was weighed 3.5gm x 3 (for three batches) and each amount was dissolved in 6ml of WFI separately, where CP (starch) was weighed 1gm, 1.4gm, 2.1gm and dissolved in 3ml of WFI respectively which was followed by oxidation at 270/4hrs. And then oxidized CP (starch) was added into Fe-salts solution to form three different batches of 0.7:1, 1:2 and 1:3 iron to polymer ratio. The Fe-nanoparticles were formulated keeping PH at 2.5 with stirring at RT (Room Temperature), 500C and 900C. The obtained ratio was used for further formulations.

Correlative development of Fe-NPs by alterations in PH and temperature

After optimizing various parameters (oxidation time, volume of oxidizing agent, iron polymer ratio) PH was considered to be an important parameter that was correlated with the heating time. PH was gradually increased by keeping temperature at RT initially which was raised to 500C by PH 3 and was maintained at same temperature till PH was reduced to 1.9 from 9 with 30min of stirring followed at each step. The temperature was evaluated till 900C for the stabilization of the Fe-iron particles was stirred for



30min after which the temperature was brought to RT gradually the Fe-particles were stirred for another 30min which was followed by ethanol precipitation, centrifugation (1000 rpm for 2min), the supernatant was removed and the sediment was redispersed into WFI. The remaining ethanol was evaporated by keeping in water bath at 500C till complete evaporation of ethanol occurs. Then the particle size was measured.

3. RESULTS AND DISCUSSIONPreformulation Study

Characterization of RMP, starch (CP), Extracted CP from RMP

PH

PH reflects a solution to be acidic or basic, PH is directly proportional to OH- concentration and inversely proportional to H+ ion concentration. The PH was used for optimization of iron nanoparticles..3.1.1.2 Particle size distribution (PSD)

The particle size was measured by particle size analyzer. The estimation of particle size was important to correlate the prepared formulations with that of RMP. The particle size analyses of the CP (starch) from material stock was done to visualize the pattern of peaks obtained.

OsmolalityOsmolality is the measure of solute concentration

in the solution being tested. It was executed to observe the effect of solute concentration. The Osmolality of the standard solution of 290 mOsm was first measured followed by RMP, same procedure was repeated for CP extracted from RMP. The observed reading was noted and it was kept for comparison with the prepared formulations and oxidized polymers.

ConductivityThe conductivity of the RMP, CP (starch) from

material stock, CP extracted from RMP was performed by using conductivity meter. Conductivity of the blank (WFI) was first taken to get exact the result of WFI which is used as dissolvent can be neglected. The results are described in Table 5. The results from the Table 5 shows the variation due to presence of free ions which are being released as conductivity is decreased in case of RMP (dialyzed) to 0.088 mS/cm when compared with conductivity of RMP (Undialyzed) which was 13.48 mS/cm. Where as conductivity of CP from RMP was due to ionization of Carboxylic group.

Polymer contentThe polymer content was calculated by steps

involving removals of excessive salts if any by dialysis, the PH of external WFI was noted for the time period till it stabilized and also the PH of RMP was noted before dialysis and after dialysis. The

dialyzed RMP was then lyophilized, the weight of dried RMP was taken and compared with the given value of iron as per product leaflet of RMP, and similarly undialyzed RMP was also lyophilized. Thus from the value obtained the amount of polymer was determined.

The process of dialysis was stopped after the stabilization of the PH of WFI which was observed after 6 hrs. Comparing the PH of RMP with that of external WFI PH it was observed that there was presence of salts in the RMP, and helped to find out the iron to polymer ratio for preparation of further formulations.

Extraction/Separation of CP (RMP)The extraction of polymer from RMP includes the

treatment of RMP with 30% HCl and ethanol, followed by dispersion into WFI. The PH and the osmolality of the extracted polymer were determined. The PH of the CP extracted from the RMP was found the RMP was found out to be 4.65. The result from above table was used for comparing the PH of the in- house oxidized polymer. Thus the effect of oxidation on PH will be utilized and compared with that of CP extracted from RMP and can be utilized for using effectively oxidized polymer for Fe-nanoparticles. Osmolality of the extracted polymer (CP) was found to be 152 mOsm. The observed value from the Table 10 was compared with the osmolality of the in-house oxidized polymer which was utilized effectively for the production of Fe-nanoparticles.

Fourier Transform Infra-Red Spectroscopic Studies (FTIR)

As oxidation of CP with NaOCl lead to formation of primary alcohols into carboxylic acid groups. In order to find out the occurrence oxidation on sample a FTIR study was performed in the wavelength range 4000/cm to 400/cm. The FTIR of the CP (starch) from material stock, CP extracted from RMP, RMP was taken as to compare whether the oxidation has occurred, spectra of unoxidised polymer was taken to observe the oxidation effect on the functional group and it was used for comparison with that of oxidized polymer (starch). The above data was used as reference standard for the formulations. The FTIR spectra of CP (starch) from material stock, CP extracted from RMP. Figure 3, 4 & 5 shows the FTIR spectra of RMP, unoxidised CP (starch) (material stock) and CP extracted from RMP. The interpretation of infrared spectra involves the correlation of absorption bands in the spectrum of an unknown compound with the known absorption frequencies for types of bonds. Thus in order to confirm the presence and amount of oxidation on to carbohydrate polymer above experiment conducted.

Now due to oxidation process the primary alcohols



of CP are converted into carboxylic acid moieties, so



the identification of the source of an absorption band are intensity (weak, medium or strong), shape (broad or sharp), and position (cmˉ1) in the spectrum. The obtained results are shown in the Table 11.

Characterization of Fecl3.6H2O (Iron salts)PH

Measurements were taken 3 times and reading was given in the Table 12.

The data from the above table was useful in comparison of readings of RMP. The PH of FeCl3 is1.22 which is highly acidic and can bring certain changes in the characteristics of the CP (starch) from material stock thus give an idea regarding the addition of polymer at PH closer to precipitation of iron that was in between 2.5-3. Thus neither the polymer will be affected nor sudden nucleation will take place and also provides with information regarding the quantity and concentration of alkylating agent to be added to raise the PH thus preventing larger dilution and sudden raise in PH can also be optimized once we know the exact amount of alkylating agent to be incorporated that will be adjusted by considering the above result as reference.

Oxidation of carbohydrate polymer (starch)Effect of NaOCl concentration on the

oxidation of starch (CP)The variation of NaOCl volume ranging from 0µl-

1000µl were used. Out of which 250µl, 270µl, 300µl and 500µl showed PH in range of the CP extracted from RMP (4.65). This experiment was performed by taking reference from effect of time on oxidation of starch (CP) regarding measurement of PH after 4hrs of oxidation. Thus the above mentioned NaOCl concentrations were used for formulation development of Fe-nanoparticles. The value of NaOCl concentration which were found out to be optimum for proper oxidation were in range of 250µl to 500µl for required oxidation of carbohydrate polymer which when compared with the PH obtained from extracted polymer from RMP, which was found out to be 4.65. The above data was used for design of further experiments.

Effect of PH on Oxidation of starch (CP)This experiment was conducted by taking 270µl

of NaOCl oxidized for 4 hours as it showed closest PH after oxidation and conductivity of 270/4 as it was found out to be closest to the conductivity of CP extracted from RMP. The effect of PH on the oxidation intensity was calculated by adjusting the PH

of oxidized CP to acidic (PH=4), neutral (PH=7) and basic (PH=9.5) with optimum PH. The optimum adjusted PH for oxidation was considered to be (9.5) as the end PH brought after oxidation was 4.56 which compared with the PH of CP extracted from RMP (4.65) was found closest and was considered optimum for further formulation development.

Effect of time on Oxidation of starch (CP)The effect of time on oxidation was found out by

determining the PH of the polymer getting oxidized at various time points with varying concentrations of NaOCl while adjusting the starting PH to 9.5 and reading thus obtained are mentioned in the Table 16.

The observed results from the Table 16 shows that 200µl to 500µl of NaOCl volume after 4hours of oxidation produces the nearer PH as that of CP extracted from RMP thus oxidation amount needed for formulation of optimized batch of Fe- nanoparticles can be correlated.

Effect of Oxidation of starch (CP) on Conductivity

Conductivity of the different concentration of NaOCl ranging from 50µl, 250µl, 1000µl, 2500µl and 5000µl of carbohydrate polymers were checked after 4hrs so as to compare the effect of oxidation process on the CP (starch) from material stock which will in relatively affect the ionization. The recorded data in Table 17. According to the observation results from the Table 17, 250µl-500µl oxidized for 4hrs can be utilized for formulation of Fe-nanoparticles. From the Figure 6, 7 & 8 shows the FTIR spectra of RMP, CP oxidized 270/240 and CP oxidized 500/240. The interpretation of infrared spectra involves the correlation of absorption bands in the spectrum of an unknown compound with the known absorption frequencies for types of bonds. Thus in order to confirm the presence and amount of oxidation on to carbohydrate polymer above experiment was conducted. The above spectra shows that CP oxidized 270/240 shows the closest resemblance of absorbance bands when compared with CP from RMP. Now due to oxidation process the primary alcohols of CP are converted into carboxylic acid moieties, so the identification of the source of an absorption band are intensity (weak, medium or strong), shape (broad or sharp), and position (cmˉ1) in the spectrum. The obtained results are shown in Table 18.

Preparation of starch coated Fe-Nanoparticles using oxidized polymer (starch) at variable NaOCl concentration

In preformulation study effect of various concentrations of NaOCl on carbohydrate polymer were screened out to compare the PH of oxidized polymer with extracted CP from RMP. The result obtained by varying NaOCl volume 250µl, 270µl, 350µl and 500µl were considered. Now to fine the NaOCl concentration and time of oxidation of starch, oxidized starch was withdrawn at different time intervals and subjected to preparation of coated Fe- nanoparticles. The observed results from the Table 19 different variable concentrations of NaOCl were utilized with time of oxidation and temperature of heating of formulation were kept at PH 2.5 and



maintained at 500C for 1hr. It was observed that 270/1 hr (conc.of NaOCl) produced the lowest particle size. But multiple peaks were observed in all the concentrations.

The observed results from the Table 20 variable concentrations of NaOCl were utilized with oxidation time and temperature of heating and formulations were kept at PH 2.5 and maintained at 500C for 1 hr. It was observed that 270/2 hrs (conc.of NaOCl) produced lowest particle size even through multiple peaks were observed. But stability at higher temp has yet to be seen. The observed results from the Table 21 variable concentrations of NaOCl were utilized with oxidation time and temperature of heating and formulations were kept at PH 2.5 and maintained at 500C for 1 hr. It was observed that 270/3 hrs (conc.of NaOCl) produced the lowest particle size. Multiple peaks were visible, due to improper coating. But stability at higher temp has yet to be seen.

The observed results from the Table 22 variable concentrations of NaOCl were utilized with oxidation time and temperature of heating and formulations were kept at PH 2.5 and it was maintained at 500C for 1 hr. It was observed that 270/4 hrs and 250/4 hrs (volume of NaOCl/Time in hrs) produced lower particle size but 270/4 hrs showed single peak where as multiple peaks were visible in all other concentrations due to unstable coating. Thus from the above data 270/4 hrs was selected for further formulation. But stability at higher temperature has yet to be observed.

Preparation of starch coated Fe-Nanoparticles using different Iron to polymer (starch) ratio

Fe-NPs were prepared by keeping PH at 2.5 and stirring at RT, 500C and 900C, with oxidized polymer containing 270µl of NaOCl volume and 240/4 hrs stirring, different iron to polymer (starch) ratio were utilized for optimization of exact ratio for the formulation of Fe-NPs. The result mentioned in Figure 12. The observed results from the Table 23 it indicates the iron: polymer (starch) ratio that was found out to be optimum as compared to other ratio, when stirred at RT was found out to be 1: 2 with particle size of 11.70nm, presence of multiple peaks can be seen in all formulations. The presence of multiple peaks were due to presence of uncoated polymer in the solution as the iron getting precipitated is very low at this PH thus the polymer amount in solution was more as compared to polymer being coated. The observed results from the Table 24 it shows that iron: polymer ratio at 500C was found out to be 1:2 with particle size of 11.70nm, presence of single peak is only seen in 0.7: 1 ratio but particle size tends to be 63.82nm which is higher than the earlier ratio, multiple peaks is basically seen due to excess of polymer as amount of iron getting

precipitated at this PH tends to be low. Stability at higher temperature has yet to be optimized for getting optimum Iron: polymer ratio.The observed results from the Table 25 it indicates

that iron: polymer (starch) ratio for Fe-NPs would be 1:2 as the Fe-nanoparticles obtained by using this ratio gave particle size of 13.54nm at 900C as compared to ratio 0.7: 1 showing the maximum particle size of 5560nm followed by ratio 1:3 which shows 68.06nm, thus 1:2 (Iron: polymer) ratio tends to show better particle size even at elevated temperature. At PH 2.5 complete precipitation of iron does not take place and thus uncoated polymer tends to show peak causing visibility of multiple peaks. Further formulation development is required to be tested at variable PH ranges.

Correlative development of Fe-NPs by alterations in PH and Temperature

The formulation of Fe-nanoparticles were performed by gradually increasing the PH of the formulation with subjected it to heating at 500C followed by gradually decreasing the PH with heating is at elevated temperature of 900C, which was followed by ethanol precipitation of the Fe- nanoparticles, centrifugation and redispersing in WFI, then increasing the PH till 5.0 and particle size were observed. The PH was adjusted with Na2CO3 till PH 2.5, the PH was further adjusted with NaOH and PH

was reduced with HCl. The observed results from the Figure 16 it shows the decrease in particle size with increase in PH from 2.5, while temperature was increased from room temperature (RT) to 500C which was maintained throughout the experiment, stirring for 30min at each step was performed.The observed results from the Table 26 it shows that

lower particle size at PH 2.5 (13.54nm) was increased to 3580nm at PH 3.5 but a decrease in particle size at PH 9.0 (615nm) was observed with heating at 500C and continuous stirring of 30min at each step which allowed the gradual coating of polymer on the precipitating Fe particles in a similar fashion. The PH

at 9.0 was gradually decreased to PH 1.9 with continuous stirring at 500C, which was heated till 900C after stirring for 30min at PH 1.9, the iron coated polymer particles where then cooled down to RT (room temperature) followed by ethanol precipitation, centrifugation, decanting the supernatant and then redispersing in WFI which was then subjected to increase in PH till 5.0 and particle size was measured.

The observed results from the Table 27 it shows that particle size reduces from 615nm at PH 9.0, to 70.28nm at PH 1.9, the particles were the subjected to higher temperature for stability of the Fe-polymer particles as multiple peaks can be seen at PH 1.9 at



500C and initially at 900C but it is not visible after



heating at 900C which was followed by purification process. The PH of the formulation was then increased till PH 5.5 for making it suitable for parenteral deliver and particle size was observed which was found out to be 40.21nm. Thus comparison of the particles size of formulated preparation was compared with that of the RMP and

it was found out to in range of 25nm-45nm. The Osmolality and conductivity were observed to be 145 mOsm and 0.4 mS/cm respectively and were compared with the above observation. The prepared formulation was subjected to autoclaving and it was found out to be stable.

TABLE AND FIGURESTable 1: PH of RMP, CP (Unoxidised), CP from RMP

Samples PH

RMP 5.46(starch) CP (Unoxidised) 5.43

CP from RMP 4.65

Table 2: PSD of RMP by DLS

Sample SizeRMP 40±0.1 nm

Table 3: Size distribution of CP unoxidised (starch) by DLS

Diameter (nm) %intensity6.608 61.2213.6 28.94113 9.9

Table 4: Osmolality of the RMP, CP extracted from RMPSample Osmolality (mOsm)

Standard solution for equipment 290RMP 380

CP from RMP 152

Table 5: Conductivity of CP from material, CP extracted from RMP, RMP and dialyzed RMPSample Conductivity (mS/cm)

WFI (blank) 0.003CP (starch) (Unoxidised) 0.119

RMP (Undialyzed) 13.48RMP (dialyzed) 0.088CP from RMP 0.418

Table 6: PH of WFI

PH of WFI Duration of Dialysis (hrs)

5.43 05.14 15.26 25.30 35.37 45.42 55.42 6



Table 7: Polymer content RMP (Undialyzed)

Weight of dried RMP (1ml) Dialyzed (a)

Iron content in 1ml of RMP (b) Polymer Content (a-b)

140mg 50mg 90mg

Table 8: Polymer content RMP (Undialyzed)

Weight of dried RMP (1ml)Undialyzed (a)

Iron content in 1ml of RMP (b) Polymer Content (a-b)

190mg 50mg 140mg

Table 9: PH of extracted polymer from RMPSample PH

CP (RMP) 4.65

Table 10: Osmolality of extracted polymer from RMPSample Osmolality

Reference solution 290 mOsmCP (RMP) 152 mOsm

Table 11: Interpretative data of FTIR for Figure 3, 4, 5

S.no Bond Compound Type Frequency range (cmˉ1)1. O-H Primary alcohols 2500-22002. C=O Carboxylic acid 1800-14003. O-H Carboxylic acid 3600-3100

Table 12: PH of the iron saltsSample PH Final PH

Reading 1 Reading 2 Reading 3FeCl3 1.21 1.25 1.21 1.22

Table 13. Calibration curve of Fe in HCL solution (λmax=340nm)

Concentration of FeCl3 (µg/ml)

Aliquot (µl)

30% HCl(µl)

Trail 1 OD @ 340nm

Trail 2 OD @ 340nm

Trail 3 OD @ 340nm

Average OD

10 10 990 0.107 0.109 0.104 0.10720 20 980 0.207 0.211 0.202 0.20730 30 970 0.299 0.297 0.301 0.29940 40 960 0.399 0.401 0.408 0.40350 50 950 0.488 0.497 0.492 0.49260 60 940 0.575 0.581 0.587 0.58170 70 930 0.642 0.648 0.663 0.65180 80 920 0.789 0.775 0.765 0.77690 90 910 0.865 0.866 0.859 0.863100 100 900 0.947 0.938 0.944 0.943

IJAPR / July 2014/ Vol. 5 /Issue. 7 / 332– 347341


Table 14: Effect of NaOCl concentration on oxidation of the polymerS.NO CP

(gm/ml)NaOCl

(µl)Initial PH Adjusted PH 10

With NaOHPH after 2 hours

Incubation

PH of oxidized

(starch) CP (4 hrs)

1 1 0 4.55 9.52 9.25 7.322 1 50 5.25 9.56 8.93 7.863 1 100 4.82 9.53 8.63 6.654 1 250 5.36 9.54 8.25 4.535 1 270 5.38 9.55 7.56 4.596 1 350 5.5 9.23 7.8 4.497 1 500 4.92 9.52 6.23 4.238 1 1000 5.42 9.52 4.30 4.189 1 2500 4.85 9.46 4.17 4.1210 1 5000 5.12 9.50 4.08 4.05

Table 15: Effect of PH on oxidation of CPCarbohydrate

PolymerVolume of NaOCl (µl)

PH adjusted (30%HCl/0.5N

NaOH)

PH after 1 hour Incubation (RT)

PH of purified CP after 4hrs

(RT)Sample 1 270 4.0 4.26 5.84Sample 2 270 7.0 6.68 6.23Sample 3 270 9.5 7.56 4.56

Table 16: Effect of time on oxidation of Starch (CP)S.NO (starch) CP

(gm/ml)Volume of NaOCl (µl)

Initial PH

Adjusted PH

PH Measured at different intervals1 hr 2 hrs 4 hrs 8 hrs 24 hrs

1 1 0 4.55 9.52 9.25 9.25 9.25 9.25 9.252 1 50 5.25 9.56 9.41 8.93 8.15 8.15 8.153 1 100 4.82 9.53 9.12 8.63 7.65 7.65 7.654 1 250 5.36 9.54 8.94 8.25 4.53 4.16 4.145 1 270 5.38 9.55 8.61 7.56 4.59 4.21 4.06 1 500 4.92 9.52 7.65 6.23 5.37 5.34 5.357 1 1000 5.42 9.52 7.12 4.30 4.28 4.26 4.25

Table 17: Effect of oxidation of polymer on conductivityS.NO (starch) CP

(100mg/5ml)Volume of NaOCl (µl)

Conductivity (mS/cm)

1 100 50 0.132 100 250 3.223 100 270 3.934 100 500 5.115 100 1000 0.786 100 2500 1.297 100 5000 2.0



Table 18: Interpretative data of FTIR for Figure 6, 7, 8S.NO Bond Compound Type Frequency range

(cmˉ1)1 O-H Primary alcohols 2500-22002 C=O Carboxylic acid 1800-14003 O-H Carboxylic acid 3600-3100

Table 19: Particle size data of Fe-NPs of different conc. PH 2.5/500C/1 hr

Batch. No Colour Volume of NaOCl

(µl)

Duration of polymer

Oxidation(hrs)

Temperature (oC)

Particle Size (nm)

Peaks

1 Black 200 1 50 28.21 Multiple2 Red 250 1 50 37.84 Multiple3 Green 270 1 50 21.04 Multiple4 Pink 300 1 50 60 Multiple5 Blue 450 1 50 68.5 Multiple

Table 20: Particle size data of Fe-NPs of different conc. PH 2.5/500C/2hrBatch. No Colour Volume of

NaOCl (µl)

Duration of polymer

Oxidation(hrs)

Temperature (oC)

Particle Size (nm)

Peaks

1 Black 135 2 50 58.77 Single2 Blue 250 2 50 43.82 Single3 Red 270 2 50 28.21 Multiple4 Green 300 2 50 50.57 Single5 Pink 450 2 50 68.05 Single

Table 21: Particle size data of Iron NPs of different conc. PH 2.5/500C/3hrBatch. No Colour Volume of

NaOCl (µl)

Duration of polymer

Oxidation(hrs)

Temperature (oC)

Particle Size (nm)

Peaks

1 Black 135 3 50 164.2 Multiple2 Blue 250 3 50 43.77 Single3 Red 270 3 50 21.04 Multiple4 Green 300 3 50 58.77 Multiple5 Pink 450 3 50 75.82 Multiple

Table 22: Particle size data of Fe-NPs of different conc. PH 2.5/500C/4 hrBatch. No Colour Volume of

NaOCl (µl)

Duration of polymer

Oxidation (hrs)

Temperature (oC)

Particle Size (nm)

Peaks

1 Green 135 4 50 32.67 Multiple2 Black 250 4 50 24.36 Single3 Pink 270 4 50 11.7 Single4 Blue 300 4 50 68.06 Multiple5 Red 450 4 50 18.17 Multiple



Batch. No colour Iron: Polymer PH Temperature (oC)

ParticleSize (nm)

Peaks

1 Green 0.7: 1 2.5 RT 21.04 Multiple2 Red 1: 2 2.5 RT 11.07 Multiple3 Blue 1: 3 2.5 RT 13.54 Multiple

Table 23: PSD of Fe-NPs of different iron: polymer ratio at RT

Batch. No colour Iron: Polymer PH Temperature (oC)

Particle Size(nm)

Peaks

1 Blue 0.7: 1 2.5 50 63.82 Single2 Green 1: 2 2.5 50 11.07 Multiple3 Red 1: 3 2.5 50 28.21 Multiple

Table 24: PSD of Fe-NPs with varying iron: polymer ratio at 500C

Batch.No

colour Iron: Polymer PH Temperature(oC)

Particle Size(nm)

Peaks

1 Blue 0.7: 1 2.5 90 5560 Multiple2 Red 1: 2 2.5 90 13.54 Multiple3 Green 1: 3 2.5 90 68.06 Multiple

Table 25: PSD of Fe-NPs of different iron: polymer ratio at 900C

Batch. No colour PH Stirring Time (min)

Temperature (oC)

Particle Size distribution

(nm)1 Red 2.5 30 RT 132 Green 3.5 30 50 35803 Blue 4.5 30 50 26694 Black 5.5 30 50 12815 Pink 7.5 30 50 7126 Orange 9.0 30 50 615

Table 26: PSD of Fe-NPs with varying PH

Table 27: PSD of Fe-NPs with varying PH

Batch. No Colour PH Stirring Time (min)

Temperature (oC)

Particle Sizedistribution

(nm)1 Green 5.5 30 RT 40.212 Red 1.9 30 90 50.573 Blue 1.9 30 50 70.28



Figure 3: FTIR spectra of RMP

Figure 5: FTIR spectra of CP from RMP Figure 6: FTIR spectra of CP extracted from RMP

Figure 1: Particle size distribution of RMP Figure 2: Particle size distribution ofunoxidised carbohydrate polymer (starch)

Figure 4: FTIR spectra of unoxidised CP (starch)

Figure 7: FTIR spectra of starch (CP) oxidized 270/240 Figure 8: FTIR spectra of starch

(CP) oxidized 500/240

Figure 9: Particle size measurement of Fe-NPs of different conc. At PH 2.5/500C/1 hr



Figure 10: Particle size measurement of Fe- NPs of different conc. At PH 2.5/500C/2 hr



Figure 13: PSD of Fe-NPs with varying iron: polymer ratio at RT (room temperature)

Figure 15: PSD of Fe-NPs with varying iron: polymer ratio at 900C

Figure 14: PSD of Fe-NPs with varying iron: polymer ratio at 500C

Figure 16: PSD of Fe-NPs with gradually increase in PH



Figure 17: PSD of Fe-NPs with varying PH

4.CONCLUSION

Figure 18: Iron nanoparticles at 40.21 nmhttp://electroiq.com/blog/2008/02/bbiomedical-applications-

The formulated Fe-nanoparticles were found to be in size range of 40.21 nm which was similar to the desired particle size range (25nm-45nm) was comparable with RMP. Stability of the Fe- nanoparticles at different temperature scale, even upto 900C have been achieved and achievement of single peak is observed thus indicating proper polymer coating over the iron particles. The obtained formulation was also subjected for autoclaving and showed no change that was found out to be stable and optimum. Stability at PH 5 which was optimum for intravenous delivery has been also stabilized.

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