INTRODUCTION:

Parkinson's disease is a degenerative neurological condition affecting the nervous system and the body's nerve-controlled regions. Dopamine is derived from the precursor chemical levodopa^1,2.

When treating Parkinson's disease, doctors usually replace dopamine with levodopa. It works best when used to treat bradykinetic symptoms, which are a hallmark of Parkinson's disease. Patients who are receiving long-term levodopa therapy have to deal with several unwanted side effects. Most of these people report changes, toxicity, aberrant motions, or a decline in effectiveness after five years³. Levodopa and carbidopa together are used to treat Parkinson's disease symptoms. When levodopa and carbidopa are administered together, levodopa's metabolism in the circulatory system is inhibited, which increases levodopa's transit into the brain. Perhaps these drugs are helpful in the early stages of Parkinson's disease. “L-DOPA tolerance” is the state in which brain and blood DA concentrations gradually equalize. L-DOPA/carbidopa dosages must be increased and given more frequently in PD patients in order to alleviate symptoms. When DA levels rise following treatment and fall in between two sessions, this dose increase results in the "on-off phenotype." It is important to have a steady supply of DA because fluctuations in its concentration throughout treatments may result in serious adverse effects, such as dyskinesias. As the treatment wears on, these negative effects get worse and outweigh its advantages. 20% of L-DOPA/carbidopa patients experienced long-term benefits after two years, according to recent research, but more than 75% experienced significant adverse effects⁴.

To decrease the dosage of L-DOPA, to boost its bioavailability, and limit systemic side effects, it is necessary to enhance its targeting to the central nervous system (CNS) and protect it from conversion by amino acid decarboxylase in the body. Over the past several decades, researchers have investigated the application of nanoparticles (NPs) to encapsulate and deliver PD medicines specifically to the blood-brain barrier (BBB) or central nervous system (CNS). This medication delivery technology not only reduced the dosage of levodopa but also eliminated or reduced the reliance on carbidopa⁵.

The most often utilized and extensively researched lipid-based nanoparticles (NPs) includes liposomes, which are composed of a double layer of phospholipids surrounding a hydrophilic water core, micelles, which consists of a single layer of phospholipids enclosing an aqueous core, and solid lipid NPs (SLNs) that have a solid hydrophobic core. The primary challenges encountered by orally delivered liposomes includes being broken down during digestion within the gastrointestinal tract, as well as undergoing significant first pass metabolism if they manage to enter the bloodstream. Therefore, a more advanced and refined version of liposome must be developed^6,7.

Nanocochleates are cylindrical structures that are created through the arrangement of lipid bilayers resulting from the interaction between negatively charged liposomes and cationic salt, typically calcium ions. These are composed of naturally occurring lipids and exhibit greater stability under harsh environmental conditions, which is a critical necessity for many nanoparticle systems, giving it a competitive edge over other options. Encapsulation of pharmaceuticals leads to reduced toxicity, enhanced bioavailability, improved therapeutic effectiveness, thus enhancing the quality of treatment for numerous diseases⁸.

Therefore, the present work aimed to develop the nanocochleate formulation loaded levodopa with improved bioavailability and reduced amount of dose.

MATERIAL AND METHOD:

Material: The API levodopa was procured from Yucca enterprises, Wadala, Mumbai.

The chemicals such as Sunflower lecithin purchased from Vitaegen Life science Nagpur, Cholesterol and Ethanol from Lobe Chemie Pvt. Ltd. Mumbai and methanol and acetonitrile were purchased from Sigma Aldrich, Mumbai.

Methodology:

Preformulation Study^9-10:

The drug and drug-excipient interaction was carried out based on the various parameters including physicochemical properties of drug, drug interaction by FTIR,DSC,UV spectroscopy and HPLC studies.

Physicochemical Properties:

The drug was evaluated for its physical appearance, melting point and solubility.

Fourier Transform Infrared Spectroscopy:

Infrared spectra were generated with an FTIR spectrophotometer (Shimadzu -801, Japan). The spectra were produced using the KBr pellet technique and the reference standard peaks were compared to the ones to make the KBr pellets. Different combination of API and excipients in dried form were used to record spectra. The base line adjustment was accomplished by the base that is KBr. The scanning wavelength range was employed from 4000 cm^-1 to 400 cm^-1resolution.

DSC:

The thermal changes of free levodopa were studied on DSC25 Mettler (Perkin-Elmer). The aluminium pans were used to heat the 5mg of sample at a rate of 50 °C/min over a temperature range from 50±1°C to 300°C. Nitrogen gas purged at a rate of 30ml/min. Empty aluminium pan was used as a reference.

Spectrophotometric analysis of levodopa (UV Spectroscopy, calibration curve):

Preparation of Standard Solution:

The standard solution of 1000µg/ml was prepared by dissolving levodopa in methanol.The further dilutions were made to produce concentration ranges from 10-1000µg/ml.

Determination of Wavelength:

The Levodopa standard solution was used to conduct the spectral analysis under ultraviolet light with a wavelength range of 200-400nm.

HPLC method development and validation^11-13:

The amount of drug in the sample was ascertained using reverse-phase high-performance liquid chromatography. Agilent Tech. (1100) equipment was used for the separation, which was performed on a Fortis C18 column (100 x 4.6mm id with 2.5mm particle size) using distilled water of HPLC grade, acetonitrile, and distilled water containing 0.15 percent Ortho phosphoric acid (OPA) in a 50:30:20 v/v ratio as the stationary phase. At a preset wavelength, detection was conducted. The 0.7 ml/min flow rate was kept constant. The quantitative values were determined using the CHEMSTATION 10.1 application.

Preparation of mobile phase:

The mobile phase was made by combining distilled water, acetonitrile, and HPLC-grade distilled water in a 50:30:20v/v ratio with 0.15% orthophosphoric acid (OPA). The solvent system's pH was kept constant at 1.5. After a 15minute sonication, the material was passed through a 0.45μm membrane filter for filtration. Degassed mixtures of solvents were employed as a mobile phase.

HPLC method Development:

Using several mobile phase compositions allowed for the optimization of the RP-HPLC chromatographic parameters. The resolution, peak symmetry, and acetonitrile-distilled water with Water (0.1% of OPA): Acetonitrile: water in a ratio 98:2 v/vwere all achieved with outstanding results using these mobile phases. Based on the peak area, the quantification of peak area was carried out at the chosen wavelength. The appropriateness of the suggested approach for the suggested system was evaluated. To make sure the system was as effective as possible, a newly made standard stock solution of levodopa was used for the system suitability test. Many criteria were examined, including resolution, peak tailing, and HETP, to assess the system's suitability.

Method validation parameters:

Five different parameters of validation were carried out and studied. Namely, linearity studied with concentration range of 2 to 100μg/ml. The concentration of levodopa was plotted against the peak area and regression equation alongwith correlation coefficient was calculated. To evaluate accuracy parameter relative standard deviation was analysed with spiking method using 80, 100 and 120% concentration addition to the set concentration of levodopa. The third parameter was precision, for which repeatability and intermediate precision was analysed. Robustness parameter was evaluated by doing deliberate changes in detection wavelength (±1nm), flow rate (±0.1ml/min), and mobile phase. Singal to noise ratio was calculated to determine limit of detection and limit of quantification.

Method of Preparation of Liposomes^14-1⁷:

Formulation by ethanol injection method:

Lecithin:cholesterol and free levodopa in various ratios were taken and dissolved in a mixture of ethanol and chloroform (1:1). Drug: Lecithin ratio employed were between 1:0.5 to 1:2 and two ratios of lecithin: cholesterol were; 2:1 and 1:1. As the lipid content in the drug: lipid ratio grew, so did the volume of ethanol and chloroform. A 15-minute sonication of the mixture was conducted using an ultrasonic waterbath. Then, utilizing a syringe and a teflon-coated bead, the homogeneous solutions were rapidly added to 25ml of deionized water that had been previously stirred at 500 RPMon magnetic stirrer. Stirring was kept up until the ethanol and chloroform evaporated. Every mixture was shaken for four to eight hours. After being made, the liposomes were stabilized for at least six hours in the refrigerator.

The composition of the formulations LDNL 1, LDNL 2, LDNL 3, LDNL 4, LDNL 5, LDNL6, LDNL 7 by injection method are given in Table1.

Formulation by reverse phase evaporation method:

Preparation of lipid phase using a rotary evaporator:

Before being utilized, the sunflower lecithin, cholesterol, and levodopa (API) were added to a 100mL round-bottom flask with a long extension neck and let to sit for 30 minutes. The mixture was subsequently treated to a solvent mixture of ethanol and chloroform (1:1). The flask was submerged in a water bath that was kept at a constant temperature of 40 degrees Celsius using a Rotary Evaporator, and it was spun at a constant speed of 150 revolutions per minute. The solvent was removed from the system at a lower pressure using a vacuum pump. A thin coating was formed after the solvent combination had completely evaporated. Chloroform was used to improve the lipids' solubility and redissolved them in the organic phase. Mannitol was dissolved with the use of heat in 25 millilitres of deionized water to create the aqueous phase. The previously prepared organic phase was combined with the prepared aqueous phase. The combination was sonicated for 15 minutes in a bath-type device at an amplitude of 80% at a pulse rate of 3 seconds, followed by a 1-second pause (no pulse) (in cold condition). The blend was subjected to sonication until a uniform opalescent dispersion was achieved. To extract the organic solvent at reduced pressure ({vacuum}) at 20–25°C and 180rpm, the mixture was put on the rotary evaporator. An aqueous solution was seen in the form of a viscous gel. To get rid of the remaining solvent, an excess of deionized water was added to the suspension that had developed and it evaporated for a further 15 minutes at 20°C. The resulting liposomes were examined using an oil emulsion microscope (100X).

The composition of the formulations LDNL0.1, LDNL0.2, LDNL0.3, LDNL0.4, LDNL0.5,LDNL0.6, LDNL0.7 by reverse phase technique are given in Table1.

Characterization of L-DOPA loaded Nanoliposomes^11-
17:

The following factors—drug loading and percent entrapment efficiency—were taken into consideration when choosing the best formulation.

Particle Size analysis and poly dispersibility Indices:

Particle size and PDI were measured at 25°C using a Zetasizer device. Malvern Instruments provided the software that was used for the analysis. Samples were kept in a refrigerator at 4°C before to analysis.

Zeta potential:

Using a Malvern Zetasizer, the zeta potential of the nanoliposome formulation was ascertained. A zeta potentiometer was used to calculate the zeta potential. A temperature of 25°C was used for the experiment.

% Drug Loading:

The drug loading was ascertained using the HPLC method.Onemillilitre of the formulation (1:1) was dissolved in one millilitre of ethanol and EDTA. Deionized water was added to bring the volume up to 10 millilitres. Five minutes were spent sonicating the solution. After that, 0.45 μm filters were used to filter the resultant solution. After that, HPLC was used to examine the filtrate.

Entrapment Efficiency:

Using a Remi cooling centrifuge, the nanoliposome formulation (10 mcg/ml) was centrifuged at 4000 rpm for 18 min at 4 °C temperature in order to isolate the free drug. The L-DNC in the suspending stage and free medication on the centrifuge tube wall are found in a supernatant. Once more, the supernatant was centrifuged for 30 minutes at 15,000rpm (4°C). Consequently, a transparent mixture of pelleted nanoliposomes and supernatant was obtained. To release the medication, the pellet of nanoliposomes was first broken up into nanoliposomes using EDTA and then broken up in 100 millilitres of ethanol. The drug entrapment was used to determine the drug released. The HPLC technology was used to estimate the quantity of levodopa. The percentage efficiency of trapping was calculated as:

Percentage Entrapment Efficiency = ------ × 100

where Wc represents the quantity of drug content (entrapped) in the nanoliposomes and Wt represents the total amount of drug in the dispersion.

HRTEM:

The morphology and size of the nanoparticles were investigated using a HRTEM (Tenai G2 20 Twin, FEI Company, Netherlands). A drop of the LDNL 3 sample was placed on parafilm for preparation. The sample drop was then covered with a drop of 2% phosphotungstic acid solution, and it was left for 30 seconds. On the sample, the copper grid was positioned. Following a one-hour air drying process, the copper grids were examined under a HRTEM and photomicrographs were taken.

Nanocochleates formulation from levodopa nanoliposome^18,19:

The trapping approach was utilized to manufacture nanocochleates loaded with levodopa. Each 25 millilitre of optimized nanoliposomal suspension received a dropwise addition of the 25 microliter (0.1M) calcium chloride solution while being vortexed. The production of nanocochleates caused the suspension to become turbid instantly. The prepared nanocochleates (LDNC) suspensions were refrigerated for approximately six hours or overnight in order to stabilize them. The prepared suspension of nanocochleates was centrifuged for 15 minutes at 20,000rpm and -4°C to create a sediment of nanocochleates, which was then lyophilized and stored.

Characterization of L-DOPA-loaded Nanocochleates^20-22:

The method described in the section on the characterization of L-DOPA loaded nanoliposomes was used to evaluate the nanocochleates.

In vitro drug release of LDNL and LDNC:

The dialysis membrane approach was used to conduct an in-vitro release investigation of free Levodopa, LDNL, and LDNC¹⁵. In three separate dialysis bags, the formulation (LDNL and LDNC) equivalent to 5 mg of levodopa and free levodopa dispersion was administered (using a 12,000 Da cut-off, Sigma). On a magnetic stirrer that rotates at 100 rpm and is temperature-controlled to 37±0.5°C for a predetermined period of time, the drug-loaded dialysis bag was suspended in a beaker containing 100ml of phosphate buffer saline held at pH 7.4. After the analysis period of 0, 0.25, 0.5, 0.75, 1, 2, and 4 hours, remove a 5ml sample and replace it with a new buffer solution. After that, a 0.45μm filter was used to filter the samples. The samples were subjected to a developed HPLC technique at 281nm for drug release analysis. Using the standard curve, the drug's release rate was calculated.

XRD:

A Japanese Rigaku Micromax-007 HF equipment with an R-axis detector IV++ and Cukα radiation (40 kV, 45 mA) was used to perform the powder X-ray diffraction (XRD) investigation¹⁶. The samples were scanned at a speed of 20.00°/min at a diffraction angle of 2̚, between 5 and 80°C. During the analysis, a nickel filter was employed to exclude Cukβ radiation. The materials' crystalline structure and phase purity were ascertained using the XRD data.

FESEM:

The produced particles' morphology was illustrated through the application of field emission scanning electron microscopy (FESEM).A drop casting process was employed to prepare the samples. In a nutshell, a cover slip was created with a thin liquid layer of sample. After being exposed to gold sputtering, the sample was captured on camera using an FEI Nova NanoSEM 450, with magnifications of 8000x and 30,000x.

Stability study:

According to ICH Q1A(R2) guidelines, the stability of the improved nanoliposome and nano-cochleate was assessed. To do stability studies on the improved drug-loaded nanoparticle suspension, a freshly made suspension was placed in an amber-colored glass vial and stored at 5 degrees Celsius for a year. Samples were taken periodically during the stability research to measure the zeta potential, EE, and particle size.

RESULT AND DISCUSSION:

Preformulation studies of levodopa:

The API is evaluated for appearance, melting point, solubility. The results for appearance, melting point and solubility are solid white powder, 276-278°C, insoluble in Diethyl ether, Chloroform, Ethanol and slightly soluble in Water (warm) and Phosphate Buffer saline pH7.4 respectively and found to be as per monograph.

Fourier Transform Infra-red spectroscopy (FT-IR):

Compatibility of drug with excipients were studied by FTIR spectroscopy using KBr pellet technique. The O-H stretching, N-H stretching, C=O stretching, C-O stretching, C-H bending are observed at 3049, 3393.19, 1648.85, 1063.13, 674.08cm^-1 respectively in both API as well as in physical mixture. There was no modification in the structure of levodopa observed when FTIR of Levodopa compared with the FTIR of physical mixture. From the above interpretation data, it was concluded that there is no appearance of chemical interaction between drug and polymers in prepared nanoparticles and it confirmed the occurrence of drug as molecular dispersion in the polymeric nanoparticles.

DSC:

The characteristic peaks corresponding to other individual ingredients in physical mixture were also present in spectra of physical mixture. There were no significant shifts or broadening of the peaks observed which might correspond to chemical interaction. Hence, it was concluded that there are no incompatibility issues.

Spectrophotometric analysis of levodopa (UV Spectroscopy, calibration curve):

The solution of drug was scanned between ranges 200- 800nm. UV spectra of the drug showed maximum absorbance at 281nm. The λmax was found to be similar with reported literature and hence used in further evaluation.

HPLC method development and validation:

Optimization of RP- HPLC method^17-22:

The optimization of any chromatographic process for analysis relies on several crucial parameters, including resolution, peak form, theoretical plates, retention time, and asymmetry. Multiple factors in chromatographic methods, including the composition of the mobile phase, flow rate, and different stationary phases, were carefully adjusted and assessed to optimize the evaluation of Levodopa and accomplish all desired characteristics. The mobile phase resulted in a peak that was of exceptional quality, with clear, symmetrical, and well-defined characteristics. The water content in the sample is 0.1% of the overall organic phase (OPA). The solvent used is a mixture of acetonitrile and water, with a ratio of 98:2 volume/volume (v/v). The flow rate of the solvent is 0.7ml per minute, and the measurement is taken at a wavelength of 281nm. The retention period of Levodopa was detected at 8.85 minutes, as shown in Figure 1.

Figure 1: Chromatogram of Levodopa

Method validation parameters:

All the validation parameters were carried out as per ICH guidelines. The linear concentration range was found to be 5 to 45mcg/ml. The linearity was confirmed by the regression coefficient value which was found to be near to 1 that is 0.999. Accuracy was also under limit and the RSD was less than 2. For precision parameter 25 μg/ml concentration of levodopa was employed. RSD for repeatability was found to be 0.59. The examination of three distinct concentrations (25, 35, 45mcg/ml) of the standard solution shown commendable repeatability. The relative standard deviation (RSD) values for interday precision were determined to be 1.49%, 1.08%, and 1.28%, whereas for intraday precision, the RSD values were found to be 1.44%, 1.04%, and 0.96%. Robustness parameter was also good with RSD below 2.This data demonstrated the method's sensitivity in determining the level of levodopa. The limits of detection (LOD) and quantification (LOQ) were determined to be 0.117μg/ml and 0.3569μg/ml, respectively.

Evaluation of Nanoliposomes:

% Entrapment Efficiency and Drug loading:

The entrapment efficiencies of prepared formulations were determined by finding the concentration of free drug in the dispersion medium. The percentage entrapment efficiency and drug loading of nanoliposomes is shown below. The percentage drug encapsulation was calculated according to the standard procedure. The percentage entrapment efficiency of formulations prepared by ethanol injection method and reverse phase evaporation method were varied between 39.45 to 86.41 and 46.90 to 79.70% respectively. The entrapment efficiency was affected by nature and amount of lecithin used. The values of percent drug loading of formulations prepared by ethanol injection method were ranges from 43.18 to 85.49% whereas % drug loading of formulations prepared by reverse phase evaporation method varied from 40.03 to 82.87%. The formulation formulated by ethanol injection method (LDNL3) showed highest entrapment efficiency i.e. 85.48% compared with all the formulations. Considering the stability of formulation based on better entrapment efficiency LDNL3 was used for further studied.

Table 2: Evaluation parameters

Formulation	Average % Entrapment Efficiency	Average % Drug loading
LDNL1	64.12±2.01	67.82±0.26
LDNL2	61.33±1.09	72.97±0.47
LDNL3	86.41±1.59	85.48±1.17
LDNL4	55.04±2.83	56.71±0.22
LDNL5	49.18±0.2	51.24±0.19
LDNL6	35.41±0.33	45.21±0.56
LDNL7	39.45±0.76	43.87±0.99
LDNL 0.1	79.7±1.95	81.25±0.28
LDNL 0.2	50.99±0.29	66.38±1.23
LDNL 0.3	51.36±1.04	61.07±0.95
LDNL 0.4	76.31±1.62	82.87±1.44
LDNL 0.5	46.9±0.6	50.07±0.77
LDNL 0.6	51.49±0.46	45.32±0.43
LDNL 0.7	53.91±0.9	40.03±0.66

Particle size, zeta potential, PDI:

LNDL3 and LNDL2 were selected based on their EL and DL percentage. The value for the determination of particles size, PDIand Zeta potential of formulation LDNL3 and LDNL2 is given in table below. Polydispersity index (PDI) is an indication of the size distribution with values ranging from 0 to 9.

This evaluation of both the nanoliposomes clearly indicated that LDNL3 formulation is superior than LNDL2 as the particle size minimum is 125.7± 0.11 and poly-dispersibility indices 0.238±0.0252 and zeta potential -31.2±0.31. As smaller particle size indicates better diffusion and permeability and lesser value of polydispersity indicates narrow size distribution of particles in the dispersion.

Zeta potential measurement the physical stability of nanodispersed system and predicted for long term stability. The maximum standard limit for zeta potential is ±30mV. Both formulations LDNL3 and LDNL2 showed good zeta potential value, indicated the better stability of formulation with no agglomeration. The results indicate the repulsive force of attraction between particles. Hence prevents aggregation. Overall, LDNL3 formulation is selected based on all evaluation parameters.

Evaluation of Nanocochleates:

From the all fourteen nanoliposomal formulations LDNL3 formulation prepared by ethanol injection method showed lowest particle size, optimum zeta potential and highest entrapment efficiency. Hence, LDNL3 was selected for nanocochleates formulation. The trapping method was used to produce the nanocochleates (LDNC).

The nanocochleates was prepared by utilizing optimized nanoliposome LDNL3. The prepared nanocochleates (LDNC) was evaluated for following parameters.

Table 3: Evaluation parameters of nanocochleates

Parameters	LDNC
Particle Size	91.2± 1.52
Zeta Potential	-53.4± 1.69
%Drug Loading	85.06 ± 1.48
% Entrapment Efficiency	90.77± 1.39

The entrapment efficiency of the formulation was considered with respect to the standard curve, which are showed in acceptable values.

DSC of Formulation:

The characteristic melting endotherm of levodopa was observed at 292.24 K, while the melting endotherm of formulation containing levodopa is 138.526 K which differs with levodopa (given in figure2). The DSC spectra reveals that in formulation the drug is well trapped and thus the endotherm energy is changed.

a. DSC of Levodopa

b. DSC of Formulation

Figure 2: DSC of a. API and b. Formulation

XRD analysis:

The Levodopa exhibited a characteristic 2Ɵ (Figure3a), whereas the LDNC exhibited a predominant lack of this feature. This could be the result of a decrease in the crystallinity of the Levodopa (Figure 3b). This may be attributed to the trapping of the drug in a nanocochleate structure, which ensures the encapsulated form of the formulation.

a: XRD of Levodopa

b: XRD of Formulation

Figure 3: XRD of a: Levodopa; b: Formulation

Microscopic study of Nanoliposome and Nanocochleates by HRTEM and FESEM:

The formed nanoparticle was spherical with smooth surface, mono-dispersed pattern confirmed the slow release of the drug. The image (Fogure 4a) was distinctly clear with a layered spherical structure with adequate size for desired bioavailability, stability and low toxicity.Surface morphology revealed tubular rod structure suggesting formation of NCs (Figure 4b). Thus, FESEM study confirmed formation of NCs by electrospray technology and validated our hypothesis.

a: HRTEM of Liposomes (LDNL 3)

b: FESEM study of Nanocochleates

Figure 4: Microscopic Study of Formulations

In vitro drug release:

The in-vitro drug release studies of free LD and LNDC were carried out at pH 7.4. The drug release studies of free LD and LNDC at pH 7.4 were performed by using the dialysis membrane method the results were shown in graphical mode below.

Around 29.5% drug released in the first hour from the LDNC which was less than Free LD (43.85%). At the end of 4 hr it was around 63.77% and 98.03% from LDNC and Free LD respectively.

Figure 5: In-vitro evaluation of Free LD and LDNC

Stability Studies:

The stability tests of nanoparticulate formulations were conducted to assess the impact of formulation quality over time. This provides a comprehensive understanding of the changes in the safety and effectiveness of the created nanoparticulate. The analysis revealed that there were no notable alterations in the physicochemical properties of the nanoparticles. As a result, the formulated preparations remained stable at a temperature of 5°C. This stability was not affected by any changes in the drug or interactions between the drug and excipients over the duration of the study.

SUMMARY AND CONCLUSION:

Parkinson's disease (PD) is a neurodegenerative condition marked by the death of dopaminergic neurons in the substantia nigra pars compacta (SNpc) leading to shortage of dopamine (DA). This deficiency causes motor symptoms such as tremors, stiffness and bradykinesia. Present treatments for Parkinson's disease (PD) focus on reducing symptoms by administering levodopa (L-DOPA) orally which is a precursor of dopamine (DA). Nevertheless, the transportation of L-DOPA to the brain is ineffective, necessitating higher doses as the disease advances, which in turn leads to severe side effects such as dyskinesias. Recent research is currently concentrating on encapsulating L-DOPA into nanoparticles (NPs) made from polymers and lipids. Thus, we aimed to enhance the effectiveness of PD treatment and minimize any negative effects. Thus, we projected to formulate nanocochelates which were able to prevent the conversion of L-DOPA into DA outside the central nervous system, therefore enhancing the transportation of L-DOPA to the central nervous system.

Nanocochleates are cylindrical structures composed of negatively charged phospholipid bilayers. These bilayers are rolled up by the interaction with multivalent counter ions (Ca2+ or Zn2+) acting as bridging agents between them. Cochleates, being a particulate system exhibit distinct characteristics such as enhanced mechanical stability and improved drug protection compared to liposomes due to their solid matrix. They have greater stability due to reduced lipid oxidation. While liposome structures are disrupted by lyophilization they can preserve their structure intact. They have the ability to effectively incorporate hydrophobic medicines into the lipid bilayer of the cochleate structure. Cochleates have the ability to gradually release a medicine when they dissociate in vivo. The nanocochleate drug delivery vehicle enables effective oral administration of medications.

Table 4: Stability Study

Stability study Period	Parameters
Stability study Period	%Drug Loading	% Entrapment Efficiency	Particle Size
Initial	85.06 ± 1.48	90.77± 1.39	91.2 ± 1.52
After 1 month	85.32 ± 0.99	90.51± 1.75	91.5± 1.44
After 3 months	85.59 ± 1.23	90.39± 0.85	92.1± 1.36
After 6 months	84.98 ± 0.96	90.25± 1.22	92.3± 1.09
After 9 months	84.86 ± 1.22	89.89± 0.85	92.9± 0.82
After 12 months	84.59 ± 1.75	89.45± 1.41	93.2± 1.24

The current study focused on creating nanoliposomes, which were then utilized to generate nanocochleates formulation with improved stability and increased bioavailability. The liposomes were prepared using two regularly known preparative methods: reverse phase evaporation and ethanol injection. The study also included the creation and verification of a straightforward technique that may be used to identify and measure the real medication in a formulation well as for studying its pharmacokinetics. The formulations were assessed by examining their morphology, analyzing their particle size and zeta potential and measuring the percentage of entrapment efficiency and drug loading. The LDNL3 formulation was utilized for generation of nanocochleates based on these parameters. The evaluation parameters such as particle size, zeta potential, entrapment efficiency and drug loading percentage substantiated the formulation of efficient nanocochleates. Furthermore, the pivotal analysis (DSC, XRD, FESEM) verified the synthesis of liposomes containing a mixture of substances and the integration of surfactants into the double layer of lipids.

The developed nanocochleates showed a significant difference in encapsulation efficiency and enabled the controlled release of levodopa for 24 hrs. Therefore, we assert that we effectively developed and showcased a nanocochleates-based nanocarrier system that possesses controlled release properties, increased stability, reduced dosage, and practical uses in the field of biomedicine.

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Received on 01.05.2024 Revised on 20.09.2024

Accepted on 21.11.2024 Published on 02.05.2025

Available online from May 07, 2025

Research J. Pharmacy and Technology. 2025;18(5):1959-1967.

DOI: 10.52711/0974-360X.2025.00280

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Sr. No.	Name of Ingredients	1	2	3	4	5	6	7
1.	Levodopa	5 mg	5 mg	5 mg	5 mg	5 mg	5 mg	5 mg
2.	Sunflower lecithin	7.5 mg	5 mg	2.5 mg	10 mg	7.5 mg	5 mg	2.5 mg
3.	Cholesterol	3.75 mg	2.5mg	1.25 mg	10 mg	7.5 mg	5 mg	2.5 mg
4.	Ethanol and Chloroform (1:1)	2.5 ml 2.5 ml	3.75 ml 3.75 ml	5 ml 5 ml	6.25 ml 6.25 ml	7.5 ml 7.5 ml	8.75 ml 8.75 ml	10 ml 10 ml
5.	Deionised water	25 ml	25 ml	25 ml	25 ml	25 ml	25 ml	25 ml