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Department of Pharmaceutical chemistry, Balwantrao Chavan College of Pharmacy, Naigaon, Nanded
Superparamagnetic iron oxide nanoparticles (SPIONs) have emerged as promising nanocarriers for targeted cancer therapy owing to their magnetic responsiveness, biocompatibility, and large surface area. In the present study, a novel carboxymethyl Assam Bora rice starch (CM-ABRS) was synthesized and employed as a natural stabilizing and capping agent for SPIONs prepared via the co-precipitation method. The carboxymethylation process was optimized, yielding a maximum degree of substitution (DS) of 1.23 ± 0.01 under optimized reaction conditions. Successful carboxymethylation of ABRS was confirmed by FTIR and NMR analyses. Structural characterization by XRD verified the crystalline nature of both CM-ABRS and magnetite nanoparticles, while SEM analysis revealed the irregular granular morphology of CM-ABRS and uniform nanoscale dispersion of CM-ABRS-coated SPIONs. The optimized formulation exhibited an average particle size of 203.7 ± 1.09 nm, a polydispersity index (PDI) of 0.027 ± 0.005, and a favorable zeta potential, indicating excellent colloidal stability. Vibrating sample magnetometry (VSM) confirmed the superparamagnetic behavior of the nanoparticles, and in vitro magnetic localization studies demonstrated their efficient responsiveness to an external magnetic field. Overall, the findings demonstrate that CM-ABRS is an effective natural polymeric stabilizer for SPIONs and highlight the potential of the developed magnetic nanosystem as a promising platform for targeted anticancer drug delivery.
Conventional chemotherapy suffers from poor tumor selectivity and systemic toxicity. Superparamagnetic iron oxide nanoparticles (SPIONs) can be magnetically guided to tumor sites and provide controlled drug delivery. Natural polymers are attractive stabilizers because of their biodegradability and biocompatibility. Assam Bora rice starch (ABRS) possesses mucoadhesive and release‑retarding properties, but its native form has limited applicability as a nanoparticle stabilizer due to poor solubility and excessive swelling. Therefore, ABRS was chemically modified by carboxymethylation and evaluated as a capping agent for SPIONs. The aim of the study was to develop CM‑ABRS stabilized SPIONs and investigate their physicochemical and magnetic properties for targeted drug delivery.
Polymer synthesis: ABRS was extracted and carboxymethylated using chloroacetic acid in isopropyl alcohol under alkaline conditions.
SPION synthesis: Magnetite nanoparticles were prepared by co‑precipitation of FeCl₃ and FeSO₄ using NH₄OH (pH 11–14) at 60–65°C under nitrogen atmosphere.
Characterization: FTIR, ¹H/¹³C NMR, DSC, XRD, SEM, TEM, DLS, zeta potential, and VSM analyses were performed.
Magnetic localization: FITC‑labeled red blood cells were used to evaluate nanoparticle localization in a microcapillary under an external magnetic field.
2.1 PREPARATION OF CARBOXYMETHYL ASSAM BORA-RICE STARCH (CM-ABRS):
Figure 1. Extraction procedure of ABRS from Assam bora rice
Figure 2. Reaction scheme for the synthesis of CM-ABRS
Table 1. Physicochemical Properties of CM-ABRS Compared with Native ABRS
|
Parameter |
ABRS |
CM-ABRS |
|
Bulk density (g/cm³) |
0.387 |
0.476 |
|
Tapped density (g/cm³) |
0.525 |
0.534 |
|
Hausner ratio |
1.358 |
1.122 |
|
Carr's Index (%) |
26.34 |
10.11 |
|
Angle of repose (°) |
34.64 |
18.34 |
|
Degree of substitution |
— |
1.23 |
The carboxymethylation process improved powder flow characteristics and produced CM-ABRS with a degree of substitution of 1.23.
2.2 Formulation of pristine magnetite (SPIONs)
SPIONs were synthesized by the co-precipitation method using a 2:1 molar ratio of FeCl₃ and FeSO₄·7H₂O. Ammonium hydroxide (0.1 M, pH 11–14) was added dropwise under continuous stirring (1500 rpm) at 60–65°C. The reaction mixture was aged for 30 min under a nitrogen atmosphere until black magnetite formed. The nanoparticles were separated by magnetic decantation, washed three times with deoxygenated double-distilled water, and dried for further characterization.
2.3 PHYSICOCHEMICAL CHARACTERIZATION
Dynamic Light Scattering (DLS)
The particle size, polydispersity index (PDI), and zeta potential of CM-ABRS SPIONs were measured using a Malvern Zetasizer Nano ZS. Samples were ultrasonicated, diluted with nitrogen-purged double-distilled water, and analyzed at 25°C.
Transmission Electron Microscopy (TEM)
The morphology of CM-ABRS SPIONs was examined using HR-TEM (TECNAI G2, 200 kV). Ultrasonically dispersed samples were placed on copper grids and imaged under high vacuum.
Fourier Transform Infrared Spectroscopy (FTIR)
The interaction between CM-ABRS and magnetite was analyzed using FTIR (Shimadzu IR Affinity-1) over the 4000–400 cm⁻¹ range. Samples were prepared as KBr pellets.
X-Ray Diffraction (XRD)
The crystalline structure of pure magnetite and CM-ABRS SPIONs was characterized using a Rigaku X-ray diffractometer with Cu Kα radiation, scanning from 10°–80° (2θ).
Vibrating Sample Magnetometry (VSM)
The magnetic properties of pure magnetite and CM-ABRS SPIONs were evaluated using a VSM (LDJ9600-1) over a magnetic field range of 0–18 kOe
2.4 In vitro Magnetic Targeting of CM-ABRS SPIONs
Blood Cell Labeling: Red blood cells (RBCs) collected from healthy mice were isolated by centrifugation, washed with glucose-saline buffer, and labeled with fluorescein isothiocyanate (FITC) following the method of Hudetz et al. The labeled RBCs were adjusted to a hematocrit of 45% with normal saline and stored in citrate-phosphate-dextrose buffer until use. All animal procedures were approved by the Institutional Animal Ethics Committee, Jamia Hamdard (Approval No. 1119).
In vitro Magnetic Localization: Lyophilized CM-ABRS SPIONs were dispersed in PBS (pH 7.4) to prepare a 5% (w/v) suspension and mixed with FITC-labeled RBCs in a 1:1 (v/v) ratio. The suspension (0.8% w/v) was passed through a 500 × 500 μm square glass capillary at a flow rate of 5 μL/min using a syringe pump. A NdFeB permanent magnet (150 mT) was positioned adjacent to the capillary to generate an external magnetic field. The localization of CM-ABRS SPIONs within the capillary was monitored using a Leica DMI 6000 CS confocal microscope equipped with a CCD camera.
Figure 3. Experimental setup for in vitro magnetic localization of CM-ABRS SPIONs using a capillary–magnet system under a confocal microscope.
3.1.1 Optimization of carboxymethylation (CM-ABRS)
|
Parameter |
Optimized value |
|
Molar ratio (starch:CAA) |
1:1.5 |
|
NaOH concentration |
8 N |
|
Reaction temperature |
65°C |
|
Reaction time |
2.5 h |
|
Degree of substitution (DS) |
1.23 ± 0.01 |
The carboxymethylation process was optimized by systematically varying the reaction parameters. The optimized conditions resulted in efficient modification of Assam Bora rice starch, producing a high degree of substitution and demonstrating the suitability of the synthesized CM-ABRS for further nanoparticle formulation and biomedical applications.
The increase in DS confirmed successful introduction of carboxymethyl groups into the starch backbone.
3.1.2 FTIR and NMR analysis
FTIR spectra showed the appearance of characteristic carboxylate bands in CM‑ABRS, confirming etherification of ABRS. ¹H and ¹³C NMR spectra further verified the formation of CM‑ABRS through the appearance of new signals corresponding to carboxymethyl carbon atoms
Figure 4. FT-IR spectra of: (A) ABRS, and (B) CM-ABRS
3.1.3 Thermal analysis
DSC analysis showed that carboxymethylation modified the thermal properties of ABRS by changing the gelatinization temperatures and reducing enthalpy (ΔH), indicating decreased crystallinity and improved molecular flexibility
|
Parameter |
Value |
|
Onset temperature |
43.62°C |
|
Peak temperature |
67.24°C |
|
Enthalpy (ΔH) |
158.863 J/g |
Figure 5. DSC Thermograms of: (A) CM-ABRS, and (B) ABRS
Table 2. Comparative of powder properties of ABRS and CM-ABRS
|
Sr. No |
Parameters |
ABRS |
CM-ABRS |
|
1 |
Bulk Density(g/cm3) |
0.387 |
0.476 |
|
2 |
Hausner’s Ratio (HR) |
1.3576 |
1.122 |
|
3 |
Compressibility index (% CI) |
26.341 |
10.11 |
|
4 |
Angle of repose (θ) |
34.64 |
18.34 |
These values indicate excellent flow characteristics of the modified polymer.
3.1.5 XRD analysis
Native ABRS exhibited diffraction peaks at 15.10°, 17.67°, and 23.01° (2θ). CM‑ABRS showed reduced crystallinity, indicating structural modification of the starch matrix.
Figure 6. X-ray diffractogram of: (A) ABRS, (B) CM-ABRS
3.1.6 Swelling and solubility
At 65°C, CM‑ABRS exhibited:
The enhanced swelling behavior is advantageous for sustained drug release applications.
Table 3. Swelling Index and Solubility of ABRS and CM-ABRS
|
Temperature |
ABRS SI (g/g) |
CM-ABRS SI (g/g) |
ABRS Solubility (%) |
CM-ABRS Solubility (%) |
|
30°C |
2.80 ± 0.35 |
8.60 ± 0.54 |
1.78 ± 0.52 |
62.83 ± 0.64 |
|
55°C |
7.21 ± 0.76 |
14.03 ± 0.80 |
11.80 ± 0.45 |
70.95 ± 0.71 |
|
65°C |
11.81 ± 0.69 |
18.86 ± 0.68 |
27.47 ± 0.71 |
79.22 ± 0.74 |
|
75°C |
15.01 ± 0.78 |
21.36 ± 0.87 |
22.04 ± 0.83 |
70.96 ± 0.34 |
CM-ABRS demonstrated substantially higher swelling and aqueous solubility than native starch.
3.1.7 Rheology
Table 4. Rheological and Mucoadhesive Properties
|
Parameter |
Value |
|
Flow behavior index (n) |
0.45 |
|
Viscosity (Pa·s) |
2.92 |
|
Consistency (g·s) |
241.36 ± 1.25 |
|
Adhesiveness (g·s) |
141.11 ± 1.60 |
|
Mucoadhesion (g·s) |
6.12 ± 0.01 |
The CM-ABRS dispersion exhibited pseudoplastic non-Newtonian flow and improved gel consistency.
3.2 Synthesis of CM-ABRS-SPIONs
Table 5. Optimized CM-ABRS-SPION Formulation
|
Parameter |
Optimized Value |
|
Polymer concentration |
0.8% w/v |
|
Homogenization speed |
25,000 rpm |
|
Homogenization cycles |
3 |
|
Particle size |
203.7 ± 1.09 nm |
|
PDI |
0.027 ± 0.005 |
Effect of polymer concentration:
The optimum polymer concentration was 0.8% w/v, producing particles of 421.3 ± 5.82 nm with PDI 0.302 ± 0.013.
Effect of homogenization:
The best formulation was obtained at 25,000 rpm for 3 cycles.
Particle size: 203.7 ± 1.09 nm
PDI: 0.027 ± 0.005
The low PDI indicates a highly monodisperse nanoparticle population.
3.2.1 Formulation of CM-ABRS coated SPIONs (CM-ABRS-SPIONs)
Figure 7. (A) Aqueous dispersion of CM-ABRS SPIONs, (B) CM-ABRS SPIONs attracted by magnet and (C) Lyophilized CM-ABRS SPIONs
3.2.3 TEM and zeta potential
Figure 8. TEM micrograph of: (A) Pristine magnetite (B) CM-ABRS SPIONs
TEM images confirmed nearly spherical nanoparticles with uniform distribution. Zeta potential analysis demonstrated sufficient surface charge to maintain colloidal stability.
Figure 9. (A) Effect of homogenization cycles on particle size of CM-ABRS SPIONs
(B) Particle size distribution curve of optimized CM-ABRS SPIONs and
(C) Zeta potential distribution curve of optimized CM-ABRS SPIONs
3.2.4 Crystallographic properties of magnetite: X-Ray Diffraction
XRD analysis confirmed the cubic crystalline structure of Fe₃O₄ in both pure magnetite and CM-ABRS-SPIONs. The CM-ABRS coating preserved the crystal structure, indicating successful surface modification without affecting magnetite crystallinity.
3.2.5 Magnetic properties
VSM analysis showed superparamagnetic behavior with negligible remanence and coercivity, confirming the suitability of the nanoparticles for magnetic targeting applications.
Figure 10. VSM results showing the M–H curves for: (A) Pristine magnetite, and (B) CM-ABRS SPIONs
3.3 In vitro magnetic localization
FITC-labeled red blood cells (RBCs) were used to visualize the in vitro localization of CM-ABRS SPIONs inside a microcapillary under an external magnetic field. The SPIONs appeared as black particles against the fluorescent RBC background. Upon application of the magnetic field, the nanoparticles accumulated along the bottom wall of the capillary due to the dominance of magnetic force over the hydrodynamic drag force (Figure 19).
The extent of SPION aggregation increased progressively with time and reached a steady state after 600 s, with no further significant increase in localization. The results demonstrate the effective magnetic responsiveness and targeting capability of CM-ABRS SPIONs, highlighting their potential as carriers for targeted drug delivery. Once localized at the target site, the drug can be released from the polymeric matrix in response to physiological or enzymatic stimuli.
Figure 11. In vitro localization study of CM-ABRS SPIONs inside the micro capillary from t= 0 to 600s
4. CONCLUSION
A novel carboxymethyl Assam Bora rice starch (CM-ABRS) polymer was successfully synthesized and employed as a natural stabilizing agent for superparamagnetic iron oxide nanoparticles (SPIONs). The optimized CM-ABRS-SPIONs exhibited a nanoscale particle size (203.7 nm), excellent monodispersity (PDI 0.027), good colloidal stability, and superparamagnetic behavior. In vitro magnetic localization studies demonstrated efficient responsiveness of the nanoparticles to an external magnetic field, highlighting their potential for targeted anticancer drug delivery. The developed nanosystem combines the advantages of a biodegradable natural polymer with magnetically responsive nanoparticles and represents a promising platform for future targeted drug delivery and theranostic applications.
REFERENCES
Shivani Shettiwar, Sumera S. G., Dr. Vishal Kshirsagar, Nano-Enabled Chemical Design of Anticancer Drug-incorporated Superparamagnetic Iron Oxide Systems, Int. J. of Pharm. Sci., 2026, Vol 4, Issue 8, 1454-1466. https://doi.org/10.5281/zenodo.21843158
10.5281/zenodo.21843158