1. INTRODUCTION:

The use of medicinal plants as a treatment method is as old as human civilization and the traditional healthcare system in many cultures worldwide is based on the use of medicinal plants. The World Health Organization (WHO) notes that about 80% of the world utilizes traditional medicine as their primary healthcare services, with herbal preparations making up a large percentage of these treatment services⁴. The revival of interest in plant-based therapeutics has been facilitated by a number of factors such as the constraints and adverse impacts of synthetic drugs, the rising demand of natural products and increased scientific evidence on the efficacy of phytochemicals in the treatment of diseases².

Over recent years, drug delivery systems based on nanotechnology have become a new solution to the shortcomings of herbal preparation Of these, vesicular drug delivery systems, such as liposomes, niosomes, ethosomes and transfersomes, as well as invasomes, have become the focus of attention because they can enclose hydrophilic, lipophilic drugs, they can prevent the degradation of labile drugs and they can facilitate the penetration of drugs across biological membranes³. These nanocarriers present a number of benefits, such as enhanced drug solubility, controlled and sustained drug release, targeted drug delivery, decreased systemic toxicity and enhanced therapeutic efficacy.

Inavasomes constitute a new group of elastic vesicular drug delivery systems that have demonstrated significant potential in improving transdermal drug delivery of drugs, such as phytochemicals Initially, invasomes were developed and consists of phospholipids, ethanol and terpenes or essential oils, as penetration enhancers The distinct structure of invasomes gives them outstanding elasticity and deformability, which allow them to squeeze through the small intercellular spaces of the stratum corneum and have a greater penetration depth than traditional liposomes and niosomes. Terpenes or essential oils in the vesicular membrane also increase drug permeation by affecting the lipid organization of the stratum corneum and making the membranes more fluid.

This is a comprehensive survey that is intended to critically review the existing body of literature on the preparation, characterisation and pharmacological assessment of invasomal formulations of T. procumbens extracts¹. The review would include comprehensive discussions on the phytochemical constituents of T. procumbens, the different preparation methodologies of invasomes, the modern techniques of characterization and the pharmacological studies of these novel formulations. Moreover, this review outlines current research gaps and future directions of developing clinically viable invasomal drug delivery systems to T. procumbens with particular focus on the input of the Indian researchers and comparative world views.

2. LITERATURE REVIEW:

2.1 Tridax procumbens: Taxonomic description and folk medicine.

Tridax procumbens is an herb that is creeping, perennial and usually reaches 15-30 cm in height, procumbent or ascending stems, opposite leaves and has daisy-like flower heads with white ray florets and yellow disc florets³. The plant can survive in a tropical and subtropical climate, which is usually found in the wastelands, road sides and farms as wild plants. Leaves are ovate to lanceolate, serrate and webbed with fine hairs and flowers are carried on long peduncles and have achene-type fruits with typical pappus hairs that help to disperse the seed to the wind⁵.

2.2 Phytochemicals of Tridax procumbens:

The phytochemical studies have found that T. procumbens is a rich source of bioactive compounds of a wide range of chemical families. Significant phytochemical subcategories found in various plant parts are flavonoids, alkaloids, terpenoids, steroids, saponins, tannins, phenolic compounds and essential oils. Flavonoids are especially abundant in the leaves and luteolin and its glycosides (glucoluteolin, luteolin-7-O-glucoside) are the most common components. Other flavonoids that have been reported in T⁵. procumbens are quercetin, apigenin and their respective glycosides.

These phytochemicals have been detected by different chromatographic and spectroscopic methods. The GC-MS analysis of methanolic extracts of the leaves of T. procumbens has revealed the presence of many bioactive compounds, such as hexadecanoic acid, octadecanoic acid, stigmasterol, beta-sitosterol and other derivatives of fatty acids. The quantitative analysis of luteolin and other flavonoids in various parts of plants has been conducted using high-performance liquid chromatography (HPLC) which is useful in the standardization of these flavonoids⁹. Luteolin in T. procumbens leaves has been reported to be ranging between 0.5-2.5% w/w based on the geographical location, the season of harvesting and the extraction process used.

2.3 Pharmacological Activities of Tridax procumbens:

2.3.1 Wound Healing Activity:

The pharmacological effects of T. procumbens have been studied in depth, with one of the most well-researched being the wound healing potential. The scientific researches have given many reasons that prove the conventional application of T. procumbens in the management of wounds². In vitro scratch assays have shown that T. procumbens extracts have significant beneficial effects on cell migration and proliferation, which are among the processes involved in wound healing⁴. T. procumbens leaves ethanolic extract has been demonstrated to stimulate proliferation of the fibroblast and collagen synthesis in a dose-dependent fashion with maximum activity of the extracts being found to occur at 50-100 mug/mL.

The wound healing effectiveness of T. procumbens has also been confirmed in vivo studies using experimental animal models⁶ compared the wound healing activity of the ethanolic extract of the leaves of T. procumbens in diabetic and non-diabetic rats, using excision and incision wound models. The findings indicated that topical application of extract (5 percent w/w ointment) was very effective in accelerating wound contraction, decreasing epithelialisation time and maximising tensile strength of the healed wounds as compared to control groups. The improved wound healing process was ascribed to the existence of flavonoid, especially luteolin, which has antioxidant and anti-inflammatory effects that promote wound healing⁹.

2.3.2 Anti-inflammatory Activity:

The anti-inflammatory effect of T. procumbens has been tested by a number of in vitro and in vivo experimental models. The plant extracts were found to suppress the synthesis of the pro-inflammatory mediators such as tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), interleukin-1 beta (IL-1beta) and prostaglandin E2 (PGE2) in lipopolysaccharide (LPS)-stimulated macrophages and other inflammat The extract of the leaf of T. procumbens in methanol had a pronounced cyclooxygenase (COX) inhibitory property with IC50 of 28.5 mug/mL and 42.3 mug/mL of COX-1 and COX-2, respectively, which suggested that the extract had the potential to reduce inflammation in the body as a natural anti-inflammatory agent.

The anti-inflammatory activity of T. procumbens extracts has been confirmed in vivo in a study of carrageenan-induced paw edema and cotton pellet-induced granuloma models in rats¹⁹. The ethanolic extract when orally given at doses of 200-400mg/kg body weight caused a significant decrease in paw edema volume and the development of granuloma tissue, similar to the effect of the standard anti-inflammatory drug, indomethacin⁷. The anti-inflammatory effect has been ascribed to the luteolin and other flavonoid content that regulates a number of inflammatory signaling pathways such as the nuclear factor-kappa B (NF-kappaB) pathway and mitogen-activated protein kinase (MAPK) pathway.

2.3.3 Antioxidant Activity:

The antioxidant properties of T. procumbens have been widely studied with different in vitro tests, such as 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation decolorization, f The methanolic extract of T. procumbens leaves has been found to have strong DPPH radical scavenging properties including the ability to scavenge with IC50 values of 15-35 mug/mL, similar to the standard antioxidant ascorbic acid⁷. The total phenolic/flavonoid content of the extracts has been attributed to the high antioxidant activity.

2.3.4 Antimicrobial Activity:

Extracts of T. procumbens were shown to have a wide-range of antimicrobial properties against different pathogenic microorganisms, including Gram-positive bacteria, Gram-negative bacteria and fungi. Methanolic and ethanolic extracts of T. procumbens leaves have high inhibitory activity against Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, Pseudomonas aeruginosa and Candida albicans with minimum inhibitory concentrations (MICs) between 62.5-500 mug/mL¹⁶. The antimicrobial property has been ascribed to the existence of alkaloids, flavonoids and terpenoids that interfere with the integrity of microbial cell membranes and prevent key metabolic activities.

2.4 Vesicular Drug Delivery Systems: An Overview:

Vesicular drug delivery systems are an important breakthrough in pharmaceutical technology, which possesses distinctive benefits when it comes to the delivery of both lipophilic and hydrophilic drugs¹¹. These systems consist of bilayered vesicles which may trap drugs inside the aqueous core or lipid bi-layer, depending on the physicochemical characteristics of the drug molecule. There are great classes of vesicular drug delivery systems, such as liposomes, niosomes, ethosomes, transfersomes, phytosomes and invasomes, with their peculiarities and uses¹².

2.4.1 Liposomes:

Liposomes were initially characterized by Bangham and others in the 1960s as spherical vesicles made of phospholipid bilayers one or more of which surround an aqueous core²⁰. These vesicles have been widely explored as drug carriers because of their biocompatibility, biodegradability and capability to carry a broad spectrum of drug molecules. Nevertheless, traditional liposomes have a number of disadvantages, such as low stability, quick elimination within the bloodstream, low ability to penetrate the skin and low entrapment efficacy of some drug molecules¹⁷. Several adjustments have been made to address these limitations such as addition of cholesterol to increase stability of membranes and PEGylation to increase the circulation time.

2.4.2 Niosomes:

Niosomes are vesicles consisting of non-ionic surfactants, which have a number of benefits in comparison to liposomes, such as reduced cost, increased chemical stability and preparation³. These vesicles consist of non-ionic surfactants like alkyl ethers, alkyl esters and fatty acids, which are frequently mixed with cholesterol to increase the rigidity of the membranes. Niosomes have presented even better outcomes when it comes to the delivery of different drugs, such as herbal extracts and phytochemicals⁶. Nonetheless, similar to liposomes, conventional niosomes are susceptible to difficulties with skin penetration and permeation of biological barriers of drugs.

2.4.3 Ethosomes and Transfersomes:

Ethosomes and transfersomes are developed vesicles that have been developed specifically to deliver drugs through transdermal applications. Ethosomes are pliable, soft vesicles that consist of phospholipids, ethanol and water, although the percentage of ethanol is usually 20-45⁶. The presence of ethanol in ethosomes confers special features, such as a higher membrane fluidity, higher drug solubility and greater skin penetration. Alternatively, transfersomes are ultradeformable vesicles that include phospholipids and edge activators (e.g., sodium cholate or Tweens) and provide the vesicular membrane with outstanding elasticity ⁹.

Ethosomes and transfersomes have been shown to have a better skin penetration than traditional liposomes and niosomes. They can squeeze through the tight intercellular spaces of stratum corneum without breaking due to the elasticity of these vesicles, promoting deeper penetration of the drug into the skin layers²⁰. Nevertheless, these vesicles are not always stable under some conditions and parameters of formulation should be optimized to attain desired drug delivery properties.

2.5 Inavasomes: A New Vesicular Drug Delivery System.

The newest development in the elastic vesicular drug delivery system is called inavasomes, which incorporates both the positive features of ethosomes and transfersomes and the use of terpenes or essential oils as penetration enhancers¹⁵. The name inavasome is obtained by the Latin invasio which means the penetration or invasion due to the high ability of these vesicles to penetrate the skin. The distinctive properties of invasomes, which include phospholipids (1-5% w/v), ethanol (10-30% v/v) and terpenes or essential oils (0.5-5% w/v) give the invasomes exceptional elasticity, deformability and penetration-enhancing characteristics¹⁹.

The process of increased penetration of invasomes through the skin is multifaceted. Ethanol in invasomes reacts with the polar head groups of phospholipids in stratum corneum, which raises lipid fluidity and forms temporary pores in the skin barrier¹⁴. The terpenes or essential oils added to the vesicular membrane also boost the process of drug permeation by upsetting the lipid structure of the stratum corneum and augmenting the partitioning of drug molecules into the skin¹³. The ultradeformable character of invasomes also enables them to squeeze through the intercellular spaces of the stratum corneum, transporting encapsulated drug molecules into deeper layers of the skin.

3. METHODOLOGY:

The overall review was carried out in a systematic manner to locate, appraise and synthesize pertinent literature on the preparation, characterization and pharmacological assessment of invasomal formulations that consisted of the Tridax procumbens extracts. The review methodology used in this study is explained in the subsequent sections.

3.1 Literature Search Strategy:

The extensive literature search was performed in various electronic databases, such as PubMed/MEDLINE, Scopus, Web of Science, Google Scholar and ScienceDirect to find pertinent information on Tridax procumbens, invasomes and vesicular drug delivery systems. A combination of keywords and MeSH terms such as Tridax procumbens, in vesicular drug delivery, transdermal delivery, phytosomes, liposomes, niosomes, ethosomes, transfersomes, herbal drug delivery, nanotechnology, wound healing, anti-inflammatory and antioxidant was used as the search strategy. Search terms were combined using the Boolean operators (AND, OR) to narrow down the search results.

Only articles written in the English language and published in the last 5 years or so (a priority was given to those published in the last 3 years) were searched in order to be able to include the latest research results. Nonetheless, even seminal literature published prior to 2015 was generally included when deemed necessary to give a historical background or a background knowledge. The reference lists of identified articles were manually filtered to find more relevant publications that could have been overlooked when conducting the search of the electronic database.

The inclusion and exclusion criteria are as follows:

3.2 Inclusion and Exclusion Criteria:

The following criteria were used to include articles in this review:¹ original research articles, review articles and book chapters addressing Tridax procumbens phytochemistry, pharmacology, or drug delivery;² articles that address vesicular drug delivery system, specifically the invasomes, ethosomes, transfersomes, liposomes and niosomes;³ articles that investigate vesicular drug delivery system preparation, character.

3.3.1 Data Extraction and Synthesis:

The data were extracted using a standardized data extraction form to extract relevant data inside the articles that were included. The information that was extracted consisted of design of the study, preparation and source of Tridax procumbens extracts, phytochemical composition, method of invasome preparation and composition, characterization techniques used, pharmacological assessment techniques, major findings and conclusions. The data obtained were thematically sorted and synthesized, to give a detailed picture of what is known so far in this area.

The quality of methodology, reliability of results and validity of conclusions in the included studies were evaluated through critical appraisal of the study. The characterization techniques used were given particular attention since extensive characterization is a key to quality, safety and effectiveness of vesicular drug delivery systems. A higher weight was placed on studies that used more than one complementary characterization method in the synthesis of findings.

3.4 Quality Assessment:

Included studies were evaluated according to a number of criteria, such as:¹ methodology was clear and reproducible;² the characterization techniques used were appropriate;³ the pharmacological methods of evaluation used were valid and reliable;⁴ the statistical analysis and interpretation of results were clear and compelling; and⁵ findings and limitations were reported in a transparent way. Evidence synthesis was limited to studies of high-quality and those studies that had high methodological limitations were taken into account and their results interpreted carefully.

4. RESULTS AND THEMATIC ANALYSIS:

The literature search yielded a substantial body of research on Tridax procumbens, vesicular drug delivery systems and their applications in herbal medicine. The following sections present a thematic analysis of the key findings organized by major themes.

4.1 Preparation Methods for Inavasomes:

Invasomes have been prepared via various methods, the most widely used being the thin-film hydration method⁵. In this procedure, the phospholipids (usually phosphatidylcholine or soy lecithin) are dissolved in an organic solvent mixture (chloroform: methanol, 2:1 v/v) in a round-bottom flask. This is followed by the evaporation of the organic solvent under a low pressure in a rotary evaporator at 40-50 degrees C leading to the development of a thin lipid film on the inner surface of the flask. The dried lipid film is then hydrated with an aqueous solution with ethanol, terpenes or essential oils as well as the drug or extract to be encapsulated. The hydration process is usually performed with 40-60 degrees C and light shaking or rotating of the mixture 30-60 minutes to help the formation of vesicles.

Another ethanol injection technique is an invasive method of preparing the ethanol injection, in which an ethanolic phospholipid solution is injected into an aqueous solution with the drug or extract under constant stirring or sonication². This approach has the benefits of being simple and reproducible, but the size of the vesicles and entrapment efficiency can be less than with the thin-film hydration approach.

Sonication and extrusion methods are usually used to reduce the sizes and homogenize the prepared invasomes. Short bursts (5-15minutes) of probe sonication at the proper power levels (100-300W) can be used to effectively reduce the size of the vesicles to nanometer size (100-300nm)¹⁰. Alternatively, uniform vesicle size distribution can be obtained by extrusion through polycarbonate membranes with pore sizes that are known (100-400nm). The size reduction method used is determined by the type of encapsulated drug and the desired vesicle properties. Table 1 presents the typical composition ranges for invasomal formulations reported in the literature.

Table 1: Typical Composition of Inavasomal Formulations

Component	Concentration Range	Function
Phospholipids	1-5% (w/v)	Vesicle formation, drug encapsulation
Ethanol	10-30% (v/v)	Membrane fluidity, skin penetration
Terpenes/Essential oils	0.5-5% (v/v)	Penetration enhancement
Drug/Extract	0.5-5% (w/v)	Therapeutic agent
Water	q.s. to 100%	Aqueous phase

4.2 Characterization Techniques for Inavasomes:

The invasomes should be thoroughly characterized to guarantee quality, stability and performance. Different methods of characterisation of invasomal formulations have been applied as discussed below.

4.2.1 Particle Size/Size Distribution:

The most popular method of measuring the mean particle size, polydispersity index (PDI) and size distribution of invasomes is dynamic light scattering (DLS)⁷. Invasomes have a particle size of 100-500nm and best transdermal delivery sizes are within the 150-300nm range. The PDI values show uniformity of the size distribution of the vesicles and PDI value below 0.3 is typically considered to be acceptable in pharmaceutical use.

4.2.2 Zeta Potential:

Measurement of Zeta potential gives details regarding the charge of invasomes on their surface and is suggestive of their stability in colloidal dispersion⁸. The negative zeta potential values of inavasomes are usually between -20 and -50mV, which is due to the presence of phospholipids and terpenes. The increase in the absolute zeta potential values will mean more repulsion between vesicles caused by electrostatic interactions, which enhances their stability against aggregation.

4.2.3 Morphological Studies:

Morphology and structural features of invasomes are observed using transmission electron microscopy (TEM) and scanning electron microscopy (SEM)⁴. TEM gives high-resolution images which show the bilayered vesicular structure whereas SEM gives information on the surface morphology. Vesicular systems have also been characterized using atomic force microscopy (AFM) at a detailed surface characterization. These microscopic methods prove the existence of the spherical or near-spherical vesicles with specific bilayered structures.

4.2.4 Entrapment Efficiency:

Invasomes entrapment efficiency (EE) is calculated by distinguishing between the entrapped and non-entrapped free drug through the use of the relevant separation methods, including centrifugation, dialysis or gel filtration chromatography¹². The quantity of the entrapped drug is then determined by applying appropriate analytical procedures, e.g., UV-Vis spectrophotometry or HPLC. Inavasomes have shown excellent entrapment efficiency of a wide range of drug molecules, with an average of 60-95% entrapment efficiency depending on the lipophilicity of the drug and the formulation composition.

4.2.5 Stability Studies:

Stability experiments are done to determine the physical and chemical stability of invasomes at different storing conditions. The tests are done under the ICH guidelines, which involves accelerated stability testing at high temperatures (25 degrees C and 40 degrees C) and relative humidity (60% RH and 75% RH) in order to determine the shelf-life of the formulations¹³. The parameters that are observed during stability tests are particle size, PDI, zeta potential, entrapment efficiency and drug content. Inavasomes have been found to be stable during long periods of storage and minimal variations in physicochemical properties were observed when inavasomes were kept under the right conditions. Table 2 summarizes the typical characterization parameters and acceptable ranges for invasomal formulations.

Table 2: Characterization Parameters for Inavasomal Formulations

Parameter	Typical Range	Significance
Particle size	100-300nm	Optimal for skin penetration
PDI	< 0.3	Indicates uniform size distribution
Zeta potential	-20 to -50mV	Indicates colloidal stability
Entrapment efficiency	60-95%	Indicates drug loading capacity
Vesicle elasticity	100-300 mg/s	Indicates deformability

4.3 Pharmacological Evaluation of Tridax procumbens Inavasomes:

Although the current literature on invasomes loaded with Tridax procumbens extracts is scarce, the pharmacological analysis of invasomes loaded with the corresponding phytochemicals and herbal extracts yield valuable information on the possible use of these preparations in relation to the delivery of T. procumbens.

4.3.1 In Vitro Skin Permeation:

The permeation-enhancing properties of invasomes have been extensively studied in vitro in Franz diffusion cells using animal skin (usually rat or mouse skin) or artificial membranes to assess the permeation rates of solutes¹⁴.It has been shown that invasomes greatly improve the permeation of different drug molecules in the skin when contrasted to traditional liposomes, niosomes and ethosomes. The improvised permeation has been credited to the synergistic interaction of the ethanol, terpenes and the ultradeformability of the invasomes.

In the case of phytochemical delivery, invasomes have been demonstrated to be effective in increasing the skin penetration of flavonoids and other compounds found in plants. Lipophilic phytochemicals, such as luteolin and quercetin in T. procumbens, are good candidates to be encapsulated in invasomes and could be better absorbed by the skin and exert localized therapeutic effects¹.

4.3.2 In Vivo Pharmacological:

The improved therapeutic effectiveness of drug-loaded invasomes over traditional formulations has been shown in vivo. Better skin permeation and drug delivery and deposition with invasomes translate into the enhanced pharmacological effects in a variety of animal models¹². In wound healing uses, invasomes have demonstrated the possibility of increasing wound healing by speeding up wound healing in experimental wounds, increasing collagen production and decreasing inflammation.

The anti-inflammatory and antioxidant effects of phytochemical-loaded invasomes have been investigated in a range of experimental models and the results show improved efficacy in relation to free drug solutions or conventional formulations¹⁵. The prolonged release properties of invasomes lead to the increased duration of drug availability in the target site leading to better therapeutic effect.

4.4 Comparative Analysis: Global vs. Indian Research:

The research on Tridax procumbens and vesicular drug delivery systems has been conducted globally, with significant contributions from Indian researchers. Table 3 presents a comparative analysis of research focus and contributions from Indian and global perspectives.

Table 3: Comparative Analysis of Global vs. Indian Research Contributions

Research Area	Indian Contributions	Global Contributions
Phytochemistry	Extensive studies on traditional uses, ethnobotanical surveys, phytochemical screening	Advanced isolation and identification of bioactive compounds
Pharmacology	Wound healing, anti-inflammatory, antioxidant studies using traditional knowledge	Mechanistic studies, molecular targets, clinical trials
Drug Delivery	Emerging research on nanoformulations	Advanced vesicular systems, invasomes, transfersomes
Clinical Studies	Limited clinical trials	Regulatory frameworks, commercial products

Indian researchers have made substantial contributions to the phytochemical characterization and pharmacological evaluation of T. procumbens, with numerous studies documenting the traditional uses, ethnobotanical significance and therapeutic potential of this plant in the Indian context. The rich traditional medicine heritage of India has provided a strong foundation for scientific investigations into the medicinal properties of T. procumbens.

On the global front, research on invasomes and advanced vesicular drug delivery systems has been led by research groups in Europe, North America and Asia. The integration of these advanced drug delivery technologies with T. procumbens extracts represents an area of opportunity for collaborative research between Indian and international research groups.

5. DISCUSSION:

5.1 Critical Review of Existing Studies:

The extensive literature review indicates that much has been done in terms of the phytochemical composition, pharmacological activities and therapy of Tridax procumbens. Wound healing, anti-inflammatory, antioxidant and antimicrobial properties have been widely researched in the plant with a large body of scientific evidence backing up the traditional uses. Nonetheless, there are a number of important gaps and constraints in the existing research environment that should be discussed.

Absence of standardized extraction and formulation procedures of T. procumbens preparations is one of the significant limitations detected. The phytochemical profile of plant extracts may differ greatly based on the geographical area, the part of the plant used, time of the year and the methodology of extracting the compound⁷. This variability presents difficulties of reproducibility and consistency of therapeutic outcomes. To proceed with the preparations to clinical applications, the development of standard protocols of the extraction and formulation of T. procumbens extracts is necessary.

Although pharmacological activities of T. procumbens have been well reported in a number of in vitro and in vivo studies, the mechanisms by which the activities are carried out are not fully understood. Even though the presence of flavonoids, especially luteolin has been linked to most of the identified pharmacological effects, the exact molecular targets and signaling pathways involved are yet to be explored⁸. Further investigation on the mechanism of action of T. procumbens phytochemicals could be done by advanced molecular biology and omics techniques.

5.2.1 Vesicular Drug Delivery:

Invasomes development has been a major advancement in the technology of delivering drugs via vesicles which have a number of advantages over traditional liposomes, niosomes and even ethosomes and transfersomes. The composition of invasomes is unique and the use of terpenes or essential oils as penetration promoters overcomes the drawbacks of the previous vesicle systems in terms of obtaining efficient skin penetration⁹. The increased deformability of stratum corneum enables invasomes to penetrate the complicated structure of the stratum corneum and this leads to better drug delivery to deep layers of the skin.

Nevertheless, invasomal formulations need to be optimized based on a number of factors, such as the use of the right phospholipids, the ethanol and terpene concentration and the optimal drug-to-lipid ratios. Their interplay with each other and overall effect on vesicle properties and drug delivery efficiency are complicated and need to be investigated systematically with quality-by-design (QbD) methods¹⁰.

5.3 Research Gaps and Limitations:

Although invasomes have a great potential in the delivery of herbal drugs, a number of gaps in research have been found during this review:

First, the number of studies that specifically explore invasomes with T. procumbens extracts is lacking. Although the overall principles of the invasome technology are known, the use of this technology in T. procumbens is largely unexplored. Considering the therapeutic potential of T. procumbens and the benefits of using invasomes, this is a key area of the possible future research.

Second, the stability of invasomes (especially those that involve complex herbal extracts with several phytochemical compounds) over a long period needs to be explored further. The chemical complexity of herbal extracts presents further difficulties in ensuring the stability of formulations during prolonged storage⁸.

Third, the safety and biocompatibility of invasomes, particularly in the case of long-term usage or application on damaged skin (e.g., in wound healing), should be thoroughly considered. Although each of the constituent components of invasomes (phospholipids, ethanol, terpenes) can be considered safe, the safety profile of the full system of vesicles needs to be evaluated carefully¹.

Fourth, invasomal formulations have practical challenges associated with the scale-up and commercialization process, such as manufacturing, quality control and regulatory. These problems need to be solved by academic and industrial efforts to translate the laboratory-scale formulations into commercially viable products.

5.4 Future Research Implications:

This review has a number of implications on future research directions. The internalization of T. procumbens extracts into the complex vesicular drug delivery systems, especially invasomes, is a prospective strategy towards improving the therapeutic effects of this medicinal plant. Future research needs to be directed towards the systematic optimization of invasomal formulations in the delivery of T. procumbens, its complete characterization by a variety of complementary methods and its full pharmacological assessment in terms of appropriate in vitro and in vivo models.

The implementation of QbD principles in developing T. procumbens-loaded invasomes may help in identifying important formulation and process parameters, which will be used to develop robust and reproducible formulations. Also, the use of state-of-the-art characterization methods, including small-angle X-ray scattering (SAXS) and cryo-electron microscopy (cryo-EM), have potential to give further understanding of the structure of invasomes and their connection with drug delivery efficacy.

6. FUTURE RESEARCH DIRECTIONS:

Following the intensive review of the existing literature, there are various future research directions that were identified that could be used to further develop and translate Tridax procumbens extracts into invasomal formulations into clinical applications.

6.1 Development and Optimization of Formulations:

Future studies ought to aim towards systematic formulation and optimization of invasomal formulations specifically suited in T. procumbens extracts. This involves the choice of the correct types and concentrations of phospholipids, optimization of ethanol and terpenes and the establishment of the best drug-lipid ratios. Experimental design methods, including response surface methodology (RSM) or artificial neural networks (ANN), might help to determine the optimal formulation factors with the least number of experimental tests¹⁷.

The investigation of different terpenes and essential oils as penetration enhancers in invasomes represents another area of interest. Different terpenes, such as limonene, eucalyptol, menthol and citral have shown penetration-enhancing properties and their relative assessment of invasomal formulations may offer useful information on the optimisation of formulations¹.

6.2 Advanced Characterization Studies:

The quality and performance of invasomal formulations rely heavily on comprehensive characterization of the formulations with advanced analytical techniques. To gain a comprehensive insight into the nature of vesicles, future research must include most of the complementary characterization methods, such as DLS, zeta potential measurement, TEM, SEM, AFM and X-ray diffraction (XRD)⁵.

The use of high-order spectroscopic methods, i.e. fourier- transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (NMR) spectroscopy, may lead to the understanding of the molecular interactions of the T. procumbens phytochemicals with the vesicular components. Also, confocal laser scanning microscopy (CLSM) might allow visualizing the penetration of vesicles and the distribution of drugs in the layers of the skin.

6.3 Pharmacological Evaluation (in vitro and in vivo):

To determine the therapeutic efficacy of T. procumbens-loaded invasomes, it is important to thoroughly pharmacologically assess these particles using pertinent in vitro and in vivo models. In vitro experiments used in wound healing purposes should comprise cell viability tests, cell migration tests (scratch assays) and collagen synthesis tests with human dermal fibroblasts and keratinocytes¹¹. Parameters that need to be assessed in vivo experimentally using standardized wound healing models in experimental animals include wound contraction rate, epithelialization time, tensile strength of healed wounds and histological analysis of wound tissue.

6.4 Safety and Toxicity Studies:

Comprehensive safety evaluation of T. procumbens-loaded invasomes is essential before clinical application. Acute and sub-chronic toxicity studies should be conducted according to OECD guidelines to establish the safety profile of these formulations¹⁶. Skin irritation and sensitization studies using animal models or reconstructed human epidermis models should be performed to assess the local tolerance of invasomal formulations.

The biocompatibility of invasomal components, particularly the terpenes or essential oils used as penetration enhancers, requires thorough evaluation. While many terpenes are derived from natural sources and are generally regarded as safe, their safety profile when incorporated into vesicular systems and applied to compromised skin needs careful assessment.

6.5 Clinical Translation and Regulatory Considerations:

The ultimate goal of developing T. procumbens-loaded invasomes is their clinical application for wound healing and other therapeutic indications. Future research should address the regulatory requirements for the approval of herbal drug-loaded nanovesicular formulations, including quality control standards, stability testing protocols and clinical trial design considerations.

Collaborative efforts between academic researchers, pharmaceutical industry partners and regulatory agencies could facilitate the translation of promising laboratory findings into clinically viable products. The development of standardized protocols for the preparation, characterization and evaluation of herbal drug-loaded invasomes would contribute to the advancement of this field.

6.6 Integration with Modern Technologies:

The integration of invasomal drug delivery with modern technologies, such as 3D printing for personalized wound dressings, smart materials for responsive drug release and combination therapies with other bioactive agents, represents exciting opportunities for future research¹⁹. The development of multifunctional wound healing systems that combine the therapeutic benefits of T. procumbens with advanced drug delivery and monitoring capabilities could revolutionize wound care practices.

7. CONCLUSION:

The present review is a critical summary of the preparation, characterization and pharmacological analysis of invasomal preparations of extracts of the plant, Tridax procumbens, which has a strong therapeutic potential because of its rich phytochemical profile (flavonoids, alkaloids, terpenoids and phenolics) and a wide range of activities such as wound healing, anti-inflammatory, antioxid Advanced elastic vesicles, invasomes, which are comprised of phospholipids, ethanol and terpenes or essential oils, provide greater deformability and skin penetration and are highly useful in transdermal delivery of phytoconstituents of *T. procumbens* and overcomes formulation constraints in standard formulations. Future research directions outlined in the review include optimization of formulations, further characterization, in vitro and in vivo pharmacological testing and safety testing and the combination of quality-by-design strategies. It also recognizes the great contributions of the Indian researchers and emphasizes the significance of the joint efforts to integrate the current delivery technologies with the traditional medicinal knowledge and eventually contribute to the creation of safe and effective therapeutic methods with the use of this promising medicinal plant.

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Received on 19.05.2026 Revised on 10.06.2026

Accepted on 30.06.2026 Published on 07.07.2026

Available online from July 10, 2026

Res. J. Pharma. Dosage Forms and Tech.2026; 18(3):217-226.

DOI: 10.52711/0975-4377.2026.00032

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