INTRODUCTION

As important as planning and executing such a project is, one very important step - probably one of the most important ones! - is how will we implement it? We are, after all, producing a very important therapeutic compound which its easier access could benefit a lot of people - specially in Brazil, where our legislation is so strict. So, how can we get it to them?

That is how our team started this research. Our goal was to, firstly, understand how CBD is originally purified from C. sativa. With those results in mind, we then drew a plan regarding multiple questions we had about our final bioproduct: how can we purify it after our genetically modified yeast is done producing it? Is it bioavailable? How do the physicochemical properties of CBD influence the way we commercialize it? How to commercialize it?

A lot of questions, we know! So, let’s dive in and understand a little bit more about it.

END-USERS

CBD end-users span a wide range of medical fields due to its diverse effects on different receptors. The most prominent group includes individuals with epilepsy, particularly those with severe conditions like Dravet and Lennox-Gastaut syndromes. Clinical studies have shown that CBD can significantly reduce seizure frequency, making it a valuable anti-seizure treatment approved by both the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) (Orrin et al, 2017).

Another important category of end-users is chronic pain sufferers. Although CBD products specifically for pain management are not yet widely approved, early studies suggest its potential, especially in neuropathic pain and conditions like peripheral neuropathy. Despite some conflicting evidence, CBD is seen as a promising alternative to opioids due to its good tolerability and its impact on improving sleep and quality of life (Boyagi et al, 2020).

Finally, people dealing with anxiety and related mental health disorders represent another key group. Research has shown CBD's potential to alleviate anxiety in treatment-resistant patients, including young individuals and those suffering from social anxiety. It is also used in cases of depression and substance abuse, indicating its growing importance in mental health treatments (Laczkovics et al, 2020).

In addition to these primary areas, ongoing research suggests that CBD could benefit individuals with a variety of other conditions, including cancer, anorexia, schizophrenia, multiple sclerosis, neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease, HIV infection and autism, making its potential applications in biomedicine vast and continually expanding (Trojan et al, 2023).

CBD IN BRAZIL - DEFINING THE PROBLEM

Worldwide, cannabidiol's potential is being extensively explored due to its positive results in diseases that manifest extremely uncomfortable symptoms and can cause irreversible sequelae (Iversen, 2001). In the psychological and psychiatric scope, studies show that CBD has significant anxiolytic and antidepressant effects (Lujan Ramirez et al, 2019). More advanced studies on cannabidiol have led to its use in treating cases of epilepsy with subsequent seizures related to Lennox-Gastaut, Doose, and Dravet syndromes (Matos et al., 2017), as well as showing successful treatment of diseases ranging from inflammatory to neurodegenerative (Donk et al, 2020).

Unlike Δ⁹-tetrahydrocannabinol (Δ⁹-THC), one of the many cannabinoids present in Cannabis sativa, cannabidiol does not produce psychoactive effects (Iversen, 2001). For example, CBD is one of the main components of the anticonvulsant medication Epidiolex®, used to treat severe cases of epilepsy in children (Pellati et al, 2018).

Despite this, the medicinal use of CBD faces various impediments that hinder its dissemination to patients. According to Anvisa's RDC 327/2019, the procedures established in this resolution apply to the manufacture, importation, commercialization, monitoring, inspection, prescription, and release of industrialized products that contain cannabis-derived plant substances or phytopharmaceuticals. As a result, products containing cannabidiol are directly affected and end up requiring a lengthy and costly bureaucratic process, thereby limiting the population's access to this treatment option.

Given the current Brazilian scenario, characterized by various legal restrictions and regulations that impose difficulties both in importing and producing cannabidiol, the existence of alternatives for its acquisition is of utmost importance for people in need of this medicine. In this context, the production of this compound through synthetic biology, as proposed in this project, appears to be a promising alternative for synthesizing cannabidiol, which, until now, requires the extraction from the plant.

EXTRACTION AND PURIFICATION OF CBD: C. SATIVA VS. our method

Purification method for Cannabis sativa

When talking about CBD’s the extraction methods from Cannabis sativa, we can classify them as traditional, such as organic solvent extraction, and innovative, such as ultrasonic assisted extraction (UAE), microwave assisted extraction (MAE), pressurized solvent extraction (PLE) and supercritical fluid extraction (SFE). The first one is the most used in a pilot-industrial scale, and it involves the plant’s maceration in the solvent, the extract being concentrated by removing the solvent under reduced pressure (Lujan Ramirez et al, 2019). The organic solvent mainly adopted is ethanol (Baldino et al, 2020), but it can be selected from a variety of compounds such as acetone, acetonitrile, benzene, butanols, chloroform, cyclohexane, hexane, isopropanol, methanol, pentane, toluene or various combinations of these chemicals, much of which present high toxicity (Lujan Ramirez et al, 2019).

The problem with traditional methods of obtaining CBD involves the type of organic solvent used, particularly its chemical compatibility with the target compounds. After extraction, the organic solvent needs to be removed during downstream processing, which is costly, and solvent residues can reduce the quality of the final product. Strict limits are imposed on solvent residues, especially in pharmaceutical applications. Consequently, the presence of solvent residues, lengthy extraction times, high energy consumption, and the need for post-treatment processes to separate and purify the extracts, as well as environmental pollution, must all be taken into account when evaluating these methods as a whole. (Baldino et al, 2020).

Of the innovative methods, however, we can see that only a small number of the possible processes have been adjusted to an industrial production scale (Eggers, 2012). The reasons behind it include the cost of installation and safety concerns. Of the mentioned methods, pressurized fluids, like carbon dioxide (SFE-CO₂) are the most documented to the point of scale-up to create cannabis concentrates, with the fluid being employed in both subcritical and supercritical states. Studies conducted around SFE-CO₂, however, show that liquid CO₂ is not a selective solvent due to its high density, being capable of co-extracting unwanted compound families such as Δ⁹-THC (Baldino et al, 2020), which is psychoactive. That means that optimizing the conditions for extraction and fractionation is vital; nonetheless, it is challenging due to the absence of essential thermodynamic solubility and phase equilibrium data (Lujan Ramirez et al, 2019). On top of that, studies that show the progress with SFE-CO₂ also show that it is a process that requires many steps besides the extraction (See Figure 1), or the combined use of SFE-CO₂ with organic solvents (See Figure 2), which can turn the whole process either way too expensive or defeat the purpose of studying alternative extraction methods altogether (Marzorati et al, 2020; Moreno et al, 2020).

Ultrasonic Assisted Extraction (UAE) is an innovative method for the extraction of cannabinoids, which are lipophilic compounds that exhibit greater solubility in non-polar solvents, such as olive oil and medium-chain triglycerides. Unlike traditional extraction techniques, UAE significantly enhances both the efficiency and yield of the extraction process by employing cavitation energy. This energy effectively disrupts plant cell structures, thereby promoting the release of intracellular constituents into the solvent (Casiraghi et al, 2022).

Studies have demonstrated that following the optimization of all experimental parameters, Ultrasonic Assisted Extraction (UAE) yields results comparable to those obtained through traditional maceration techniques, commonly employed by pharmacists in the preparation of Cannabis extracts, while significantly reducing the duration of the extraction process. Furthermore, the exposure of olive oil to cavitation waves during the UAE process does not adversely affect the qualitative composition of the initial oil (Casiraghi et al, 2022). Other studies indicate that the application of ultrasound treatment (UAE) in conjunction with mechanical expression not only enhances oil yield but also preserves the oil's quality. This dual approach effectively maintains its antioxidant properties while minimizing the concentration of free fatty acids (Esposito & Piazza, 2022).

In conclusion, the extraction of CBD from Cannabis sativa involves both traditional and innovative methods, each with distinct advantages and limitations. Therefore, it becomes factual in our eyes that the usual CBD extraction processes are not ideal in terms of sustainability and even economic viability. That’s what our project envisions to improve while we propose a more environmentally sustainable way of extracting our synthetic CBD.

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Purification method for synthetic CBD

Since we are developing an intracellular bioproduct, it is vital to study how exactly would we purify it. The first step seems elementary: cellular lysis. We need to destroy our cell in order to get what’s inside, after all. But separation gets tricky - how to do it? That’s how we devised our implementation proposition - a liquid/liquid extraction system (LLE) based on non-ionic surfactant–salt–H₂O - also known as poly(ethylene glycol) (PEG)/salt system (Figure 3).

Why that system, you ask? Well, we had a few objectives in mind. Firstly, we wanted to propose a technique that was more eco-friendly than that of industrial CBD. Secondly, we were concerned about scale-up processing, which is all of what our project is about this year, and what LLE conveniently can be (Salabat; Alinoori, 2008).

LLE using aqueous two-phase systems have been continuously studied for it’s large-scale recovery and capacity for bioproduct’s purification (Salabat; Alinoori, 2008). Currently, only PEG/salt systems are known to be used in industrial applications. These systems require moderate concentrations of PEG and specific salts (such as phosphates or sulfates) to induce phase separation. From an economic perspective, aqueous two-phase systems (ATPS) offer advantages in terms of labor, energy, and investment costs. A more recent development in ATPS aims to address these challenges by using only a single phase-forming component at lower concentrations. Such systems are particularly effective for separating hydrophobic and amphiphilic molecules (Selber et al, 2003). This would be perfect for CBDA, an extremely hydrophobic molecule (Drugbank, 2022). In this context, ATPS represents a promising unit operation for the downstream processing of biomolecules.

When the process as a whole is studied, a suitable type of ATPS (polymer-salt, polymer-polymer, or alternative systems) must be selected for the process, based on the desired partitioning of the target biomolecules (Benavides; Rito-Palomares, 2008). In this regard, polymer-salt ATPS is often favored due to the low cost of the phase-forming materials, which was one of our main goals with this proposition. PEG-phosphate systems are the most commonly used in the field (Rito-Palomares, 2004). Subsequently, parameters such as PEG molecular weight, tie-line length (TLL), volume ratio (VR), and pH are preselected and assessed for the recovery of the target product by referencing the relevant phase diagram and considering the physicochemical properties of the product. This helps to determine the operating conditions for the ATPE process, ultimately achieving selective partitioning (Glyk et al, 2015, as cited in Benavides; Rito-Palomares, 2008).

One major advantage of ATPS is their ease of scale-up through the use of commercially available equipment from the chemical industry (Cunha; Aries-Barros, 2002). Studies have shown successful enzyme extractions with scale-ups as large as 40,000-fold without significant loss in yield. Factors like rapid phase equilibrium, efficient mixing, and phase separation contribute to the scalability (Glyk et al, 2015 as cited by Kula; Selber, 2002).

Finally, one of the major points that stood out for us in the plotting of this plan was cost-efficiency and eco-friendliness, particularly regarding chemical consumption and waste disposal. Recycling the phase-forming components, like PEG and salts, is not only possible but done, and can significantly reduce costs (Kula; Selber, 2002). Studies have evaluated PEG recycling in a two-stage ATPS process, incorporating phase recycling and ultrafiltration. This process involves extracting the target product into the PEG-rich phase and later recovering it in the salt-rich phase (Rito-Palomares, 2004). Recycled salt and PEG can be reused in multiple cycles, reducing fresh chemical consumption by up to 50% in some cases. Additionally, phosphate-rich phases can be recycled for up to four cycles without compromising performance, further lowering costs and environmental impact (Kula; Selber, 2002).

PHYSICOCHEMICAL PROPERTIES

In order to create any pharmaceutical drug, the first step is to understand how our molecule of interest acts. The most distinctive characteristic of CBD is its low solubility in water, at approximately 0,1 µg/mL at 37ºC. (Koch et al, 2020). This generates some difficulties in regards of their use in some pharmaceutical preparations - which is why pure CBD is typically administered in oil or alcohol based formulations (Millar et al, 2020).

According to Fraguas-Sánchez et al, 2020, CBD exhibits various sensitivities to environmental factors, such as temperature, light and presence of oxygen. In the presence of oxygen, CBD oxidizes to mono- and di-hydroquinones (Mechoulam; Hanus, 2002). The degradation kinetics of CBD are much higher in aqueous solutions, partially due to the presence of dissolved oxygen in the medium (Fraguas-Sanchéz et al, 2020). In addition to that, various research studies have noted that CBD undergoes instability when exposed to room temperatures or above, oxidizing into cannabidiol hydroxyquinone (Watanabe et al, 1991; Pacifici et al, 2017). To guarantee a longer shelf life, it is imperative to store it in the refrigerator. Cannabinoid molecules are also sensitive to light; photolytic reactions involving CBD are, too, oxidative, degrading more with high temperatures and light levels. With all of that in mind, it is almost obvious that in physiological conditions (pH 7,4 and 37 ºC) CBD would be highly unstable, with 10% of the drug being degraded in 24 hours. (Fraguas-Sanchéz et al, 2020). In Tables 1 and 2, it’s possible to verify all of those properties we just explained.

Physicochemical Properties of CBD	Value	References
Physical nature	Solid and crystalline	(Dickman and Levin 2017; Drugbank 2022)
Water solubility	0.1 µg/mL at 37°C, 0.02 µg/mL at 30°C, 5 µg/mL at 37°C	(Koch et al. 2020; Li et al. 2021; Tabboon et al. 2022)
Solubility in other solvents	Ethanol (35 mg/mL), methanol (30 mg/mL), DMF (50 mg/mL), DMSO (60 mg/mL)	(Cayman Chemical 2022)
Molecular formula	C12H30O2	(Cayman Chemical 2022)
IUPAC name	2-[(1R,6R)-6-Isopropenyl-3-methylcyclohex-2-en-1-yl]-5-pentylbenzene-1,3-diol	(National Center for Biotechnology Information 2022)
Molecular weight	314.5 g/mol	(National Center for Biotechnology Information 2022)
log P	6.1	(Drugbank 2022)
log S	-4.4	(Drugbank 2022)
pKa (strongest acid)	9.13	(Drugbank 2022)
pKa (strongest basic)	-5.7	(Drugbank 2022)
Formal charge	0	(National Center for Biotechnology Information 2022)
Hydrogen acceptor count	2	(Drugbank 2022)
Hydrogen donor count	2	(Drugbank 2022)
Stability	Stable at cold temperatures and highly unstable at room temperature	(Fraguas-Sanchéz et al. 2020)
Storage	-20 ºC or in cold temperature	(Fraguas-Sanchéz et al. 2020; Cayman Chemical 2022)

Parameters	Stability
Temperature	Unstable at room temperature (25 °C) (t95 = 117.13 days), stable at 5 °C for approximately 12 months; increasing temperature leads to the increasing instability of CBD
Presence of Oxygen	Highly sensitive to oxygen (t95 of 1.77 days)
Light Sensitivity	Highly photosensitive; stable in dark conditions; increasing light with increasing temperature leads to degradation of CBD
Physiological conditions	Unstable in pH 7.4 (37 ± 0.5 °C); t1/2 at 25 °C is 19 days, and at 37 °C is 7 days
Solvent	Highly unstable in water compared to ethanol

Therefore, the pharmaceutical development of CBD-based drugs is significantly challenged by the compound's low water solubility and sensitivity to environmental factors, such as temperature, light, and oxygen. All of these characteristics highlight the necessity of controlled conditions for storage and manipulation of CBD, as well as developing effective pharmaceutical formulations.

BIOAVAILABILITY AND ENCAPSULATION

As the physicochemical properties of CBD show, it is a challenge to successfully use it as a medicine. CBD’s bioavailability varies greatly with the way it is administered (Huestis, 2007; Martin et al, 2018), with the oral route - the preferred one by patients and drug developers - being particularly difficult (Millar et al, 2020). As mentioned, CBD is a highly lipophilic drug. Its oil/water partition coefficient (log P) is 6,3, which is considered high and, because of it, it has to be supplied in an oil or alcoholic formulation. Even so, this characteristic makes it so that, if delivered orally, CBD can precipitate in the gastrointestinal tract and result in a slow absorption rate (Helen Chan; Stewart, 1996). According to Millar et al, 2018, CBD’s oral bioavailability is estimated at 6%.

These limitations show that formulating a way to increase oral bioavailability is crucial. With that in mind, our team conducted this literature research around encapsulation of CBD, in order to formulate a better final product.

According to Millar et al, 2020, CBD is a drug classified as a Class II Biopharmaceutics Classification System (BCS). The bioavailability of Class II drugs depends on their dissolution rate, which is directly linked to their solubility. Therefore, enhancing solubility leads to better bioavailability in vivo. (Charalabidis et al, 2019). Among the strategies we can use to improve this dissolution rate is nanonisation - a method used to reduce the particle size of an active pharmaceutical ingredient to the nanometer scale. Reducing a drug's particle size increases its surface area, which in turn leads to a proportional rise in its dissolution rate. This enhances the absorption of drugs that have poor solubility (Grifoni et al, 2022).

Overall, nanoparticle formulations show an incredible reduction of oral dose in comparison to commercially available products (Lazarotto Rebelatto; Rauber; Caon, 2023). In Table 3, we show a comparison of nano-based technologies that have been reported in literature. Such nanosystems aid in preventing drugs from being deteriorated in the gastrointestinal tract, as well as helping its delivery (Grifoni et al, 2022).

Nanovectors can be categorized based on the type of nanomaterials they are made from into three main groups: polymeric, lipid-based, and inorganic nanocarriers, the first two being much more studied in terms of cannabinoid encapsulation. Polymeric nanovectors originate from either natural sources (such as polysaccharides and proteins) or synthetic polymers. Lipid-based nanovectors encompass microemulsions, nanoemulsions, vesicles, solid lipid carriers, and nanostructured lipid carriers (Bilia et al, 2012). A few graphic examples of phytocannabinoid nanoformulations can be seen in Figure 4.

Polymeric nanoparticles

Polymeric nanoparticles (NPs) have demonstrated great potential as drug delivery systems due to their ability to provide controlled release, stability under physiological conditions, and their biocompatibility and biodegradability. They are also non-immunogenic, non-inflammatory, and capable of avoiding the reticuloendothelial system (Begines et al., 2020; El-Say; El-Sawy, 2017; Soppimath et al, 2001).

Drug molecules can be incorporated into these nanoparticles either by embedding them within the polymer matrix (non-covalently) or through covalent bonding. This versatility allows for the encapsulation of a broad spectrum of hydrophilic and hydrophobic drugs, making polymeric nanoparticles ideal for co-delivery (Srinivasta Reddy; Zomer; Mantri, 2023). However, their use is somewhat restricted by the lack of consistent results when scaled up for industrial production (Begines et al, 2020). Biodegradable polymers such as poly(lactic-co-glycolic acid) (PLGA) and poly(ε-caprolactone) (PCL) are commonly used for the encapsulation of cannabinoids.

Nanoparticle Type		Advantages	Disadvantages
Polymer-based colloidal systems	Polymer micelles	- Increased solubility for cannabinoids - Drug release in a controlled manner - Prevents psychotropic effects of cannabinoids (these NPs do not cross the blood–brain barrier) - EPR effect due to small particle size (desirable in antitumor therapies)	- Low drug-loading efficiency - Low physical stability in vivo - Dependency of critical micelle concentration
	Polymer nanoparticles	- Drug release in a controlled manner (several days), particularly interesting in chronic therapies - Increased stability and protection against degradation compared to lipid systems - Site-specific drug delivery - Drug release profile can be adjusted depending on the polymer type - Preparation methods are versatile	- Polymer degradation products can be toxic - Preparation methods usually use toxic organic solvents
Lipid-based colloidal systems	Liposomes	- The amount of cannabinoid released can be modulated by the phospholipid/cholesterol ratio - Capacity for self-assembly - High biocompatibility and interaction with biological membranes	- Lower encapsulation efficiency of cannabinoids - Low stability in biological fluids - Rapid tissue distribution or short half-life - Phospholipid may undergo oxidation - High production cost - Traditional preparation methods use organic solvents - May trigger an immune response
	Self-nanoemulsifying drug delivery systems	- Ease of preparation and scale-up - Low production cost - Provide high oral bioavailability - Reduced particle size - Reduce the effect of bile salts on cannabinoid absorption - Shorter onset of action (reduce Tmax)	- Wide particle size range as nanoparticles are formed in situ - Precipitation of drug in the gastrointestinal fluid is more common
	Nanoemulsions	- More stable systems than liposomes - Enhanced drug solubility and bioavailability - Improved hydration when used topically - Low production cost - High encapsulation efficiency for cannabinoids	- Instability phenomena such as coalescence and oxidation of oils or lipids
	Nanostructured lipid carriers	- Ease of large-scale production using high-pressure homogenization - Imperfections in the lipid matrix accommodate cannabinoids easier, impacting encapsulation efficiency - Prevent particle coalescence due to the solid matrix	- Sensitive to preparation or storage parameters, leading to lipid phase transitions - Long-term stability issues upon storage
	Lipid nanocapsules	- Lipid core increases cannabinoid incorporation, while polymer coating provides stability - Efficient surface functionalization - Low energy methods can be used in preparation
Inorganic colloidal suspensions	Carbon nanotubes	- Improved mechanical properties - Penetrate cell membranes due to reduced particle size - Intrinsic spectroscopic properties allow tracking and real-time monitoring	- Expensive production - Low degradability - Toxicological issues (pulmonary complications)
	Metal nanoparticles	- Specific optical and magnetic properties (important for cancer applications) - Strong plasma absorption - Multimodal applications - Particles characterized by uniform size and shape	- Toxicological issues - Synthesis impurities can result in unstable systems

Lipid-based nanoparticles

Lipid-based colloidal particles, including liposomes, solid lipid nanoparticles (SLN), lipid nanocapsules (LNC), nanostructured lipid carriers (NLC), self-nanoemulsifying drug delivery systems (SNEDDS), and nanoemulsions, have been studied recently as carriers for lipophilic bioactive compounds (Shirodkar et al, 2019; Teixeira; Carbone; Souto, 2017). These lipid-based nanosystems for drug delivery provide several benefits over polymeric nanoparticles, such as high drug loading capacity, low toxicity, cost-effective manufacturing, and scalability. Furthermore, they are biodegradable and highly stable. However, lipid nanoparticles come with certain challenges, like drug leakage or release from the formulation due to lipid crystallization over time during storage, and the high water content in the formulation can lower the loading efficiency. The lipid-based nanocarriers mentioned above, however, have been studied and designed for the delivery of phytocannabinoids, which have demonstrated improved stability and bioavailability of the encapsulated drugs (Srinivasta Reddy; Zomer; Mantri, 2023).

Among the lipid-based nanoparticles, liposomes are among the most studied, used, and commercially approved systems for delivering drugs, nutrients, and/or genetic material into the human body (Allen; Cullis, 2013). Liposomes are capable of encapsulating both hydrophobic and hydrophilic compounds, protecting them from degradation and enabling targeted release at specific sites. This makes it possible to administer CBD orally with accurate dosing, achieving localized effects when applied dermally or systemic effects when delivered transdermally. As the first nanomedicine delivery system to move from theoretical development to clinical use, liposomes have become a well-established technology with significant clinical acceptance (Valh et al, 2020), this makes liposomes the best choice for CBD encapsulation to the best of our knowledge.

This conclusion is further reinforced by the research conducted by Blair in 2020. The primary goal of the study was to compare the bioavailability of cannabidiol (CBD) when administered as a stand-alone active ingredient versus in a liposomal formulation. The findings clearly show that liposomal CBD exhibits significantly higher bioavailability compared to the stand-alone form. This suggests that the liposomal delivery system enhances the absorption and effectiveness of CBD within the body, making it a more efficient method of administration, as it can be seen in Table 4.

ANTIMICROBIAL ACTIVITY

Finally, we reach a very important aspect of our drug of interest: its antimicrobial activity. After all, if we are proposing a genetically modified yeast that is able to produce CBD, it would completely defeat our purpose if it kills our yeast.

Thankfully, as reported by Ali et al. (2012), neither cannabidiol (CBD) nor cannabidiolic acid (CBDA) demonstrate significant antifungal properties. This finding is important as it suggests that, despite their wide range of biological activities, these compounds cannot kill our chassis.

When looking into CBD’s antibacterial activity, however, it is found in literature that it demonstrates antimicrobial effects against Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA) strains; endocannabinoids have also been shown to be effective against biofilms (Karas et al, 2020). It is also noted that CBD is more effective as a bactericide than it’s polar analogue, CBDA. This suggests that the antibacterial properties come from CBD’s resorcinyl part, and not the terpenoid or pentyl groups (Martinenghi et al, 2020). However, Gram-negative bacteria appear to be much less susceptible to CBD and CBDA’s inhibitory effects (Vu et al, 2016). This could be due to the unique structure of Gram-negative bacteria, which have lipopolysaccharides and proteins with minimal phospholipid content in the outer layer of their outer membrane. This structure is a key factor in the membrane's strong resistance to detergents. The outer membrane is impermeable to large molecules and restricts the diffusion of hydrophobic substances. Moreover, it is resistant to neutral and anionic detergents.

In addition, CBD has been shown to be safe for human keratinocyte cells, displaying no toxicity even at concentrations up to seven times higher than the minimum inhibitory concentration required for antibacterial activity (Martinenghi et al, 2020).

CONCLUSION

In conclusion, the proposed implementation strategy effectively integrates several key factors to ensure that the production and delivery of CBD reaches our end-users efficiently. By selecting eco-friendly and cost-effective extraction and purification methods, such as the aqueous two-phase system (ATPS), we aim to optimize the production of synthetic CBD while reducing environmental impact. This scalable, industrially applicable system aligns with our goal of making CBD more accessible, particularly in regions with strict regulations, like Brazil.

The careful consideration of CBD’s bioavailability, encapsulation, and physicochemical properties ensures that the final product will be both effective and safe for end-users. By utilizing advanced liposomal delivery systems, we significantly improve the bioavailability of CBD, providing more consistent therapeutic outcomes for patients. This approach is especially beneficial for treating epilepsy, chronic pain, and mental health disorders, the primary medical fields that our target end-users are from.

Ultimately, this holistic approach, from production to delivery, ensures that we not only meet the legal and practical challenges of CBD commercialization but also provide a product that is accessible, effective, and beneficial to a wide range of patients.

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