Preparation of Cannabinoid-loaded Liposomes Using Microfluidics

July 2020 | By Dr. Natalia Sannikova, Senior Formulation Scientist at Ascension Sciences


Liposome encapsulation is used to enhance drug circulation time, reduce toxicity, protect the load from oxidation and degradation, and improve the solubility of hydrophobic molecules.

Cannabinoids are chemical compounds found in the cannabis plant. There are more than 100 cannabinoids that have been isolated from cannabis [1]. Cannabidiol (CBD), tetrahydrocannabinol (THC) and cannabinol (CBN) are the most frequently studied species (Fig. 1).

Figure 1. Cannabinoid structures.

Cannabinoids are highly lipophilic with aqueous solubility as low as 2.8 mg/L at room temperature, which leads to their limited stability in aqueous systems and reduced bioavailability in oral administration [2,3]. Furthermore, cannabinoids are sensitive to light [4], pH [5] and temperature [4]. These characteristics make them good candidates for the nanoparticle delivery technology, including liposomal encapsulation [6].

Here, we report microfluidic assembly of THC- and CBD-loaded liposomes using the NanoAssemblr Benchtop instrument. The NanoAssemblr platform has previously been used for the preparation of unilamellar liposomes comprised of synthetic phospholipids and loaded with hydrophobic drug molecules [7,8]. Liposomes were loaded with cannabinoids in situ during the formation; liposome size and size distribution (PDI) were controlled by the process parameters Total Flow Rate (TFR) and Flow Rate Ratio (FRR) on the NanoAssemblr Benchtop instrument.


Experimental Design

Unilamellar liposomes are produced by controlled solvent displacement in a microfluidic mixer as illustrated in Figure 2. Organic phase (ethanolic solution of lipids) is mixed with an aqueous buffer under laminar flow conditions. Phospholipid molecules self-assemble into liposomes during mixing as polarity of the environment is increasing due to solvent displacement. Laminar flow and computer controlled injection enable reproducible fine-tuning of the conditions of liposome formation by specifying in the NanoAssemblr software, the Total Flow Rate (the sum of flow rates between the organic and aqueous phases) and the Flow Rate Ratio (the volumetric ratio of aqueous buffer to organic solvent being mixed per unit time). The hydrophobic cannabinoid (THC or CBD) was loaded in situ by dissolving it in the organic phase along with the lipids prior to injection into the microfluidic channels. 

Figure 2. Controlled solvent displacement in a microfluidic mixer. 

1) Organic (ethanol) and aqueous (PBS) phases entering the channel under laminar flow don’t mix. 

2) Microscopic features in the channel cause fluid streams to mix in a controlled fashion. 3) Intermingling of the phases increases as they continue through the mixer. 

4) Fluids emerge completely mixed.

Materials and Methods

POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and DSPE-PEG (1,2-distearoylsn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]) were purchased from Avanti® Polar Lipids, cholesterol was purchased from Sigma Aldrich.

POPC, cholesterol and PEGylated lipid at 52:45:3 molar ratio were dissolved in absolute ethanol. Calcium- (Ca2+) and magnesium- (Mg2+) free PBS buffer at pH=7.4 was used as the aqueous phase. The organic and aqueous phases were rapidly mixed using the NanoAssemblr Benchtop microfluidic instrument at different Flow Rate Ratios (1.5:1 or 2:1) and Total Flow Rates of 12 mL/min to form unilamellar liposomes of various sizes. Cannabinoids were loaded by dissolving it in the lipid solution at a concentration of 1 mg/mL. Formulations were then dialyzed against PBS for ethanol removal. At Flow Rate Ratio 1.5:1, formulations were diluted down to 10% ethanol immediately following the mixing process and before dialysis, since high amounts of ethanol can destabilize liposomes.

Particle size and integrity were measured before and after dialysis using Dynamic Light Scattering (Zetasizer, Malvern Instruments, UK). Size and polydispersity index (PDI) were represented as the mean of 3 samples, and error bars represent standard deviation of the mean. Cannabinoid content was quantified by HPLC.


Results and Discussion

Liposome size depends on formulation parameters TFR and FRR. At constant TFR, liposome size decreases asymptotically with increasing FRR; similarly, at constant FRR, size decreases with increasing TFR. Such behaviour has been previously described for synthetic liposome formulations2, and the asymptote in size has been found to correspond to the so-called “limit size” representing the smallest possible particle size based on ideal packing of constituent molecules2. We have prepared empty POPC liposomes at constant TFR of 12 mL/min and several FRR values. Our results are consistent with the published trends (Fig. 3).


Lipid composition Soy-PC:Chol:DSPE-PEG (52:45:3 mol%)
Total lipid concentration 10 mg/mL
Organic solvent Ethanol
Aqueous solvent PBS pH 7.4
Total Flow Rate 12 mL/min
Flow Rate Ratio n:1 – n indicated on horizontal axis
Solvent Removal Dialysis


Figure 3. Tuning liposome size with microfluidics. Hydrodynamic size and particle size distribution (expressed as polydispersity index, PDI) (both determined by dynamic light scattering) of POPC liposomes formulated at various total flow rates on the NanoAssemblr Benchtop instrument.

Cannabinoid-loaded liposomes were prepared at FRR of 1.5:1 and 2:1. In situ loading of  liposomes was achieved by dissolving corresponding cannabinoid (THC or CBD) and the lipid mixture at a 0.1 w/w ratio in the organic phase (Fig. 4). 


Lipid composition  Soy-PC:Chol:DSPE-PEG (52:45:3 mol%)
Total lipid concentration 10 mg/mL
Cannabinoid concentration in lipid mix 1 mg/mL
Drug/lipid ratio 0.1 w/w
Organic solvent Ethanol
Aqueous solvent PBS pH 7.4
Total Flow Rate 12 mL/min
Flow Rate Ratio n:1 – n indicated on horizontal axis
Solvent Removal Dialysis


Figure 4. Cannabinoid-loaded liposomes stability. Hydrodynamic size and particle size distribution of cannabinoid-loaded POPC liposomes formulated on the NanoAssemblr Benchtop instrument monitored for 5 weeks.

Size of the loaded liposomes increased slightly compared to the empty liposomes prepared with the same formulation parameters. We have observed minor size fluctuations for the loaded liposomes at FRR 1.5:1 and a more noticeable size change for the loaded liposomes at FRR 2:1. Loaded liposomes prepared at both FRR values have comparable sizes. At FRR 1.5:1 CBD-loaded liposomes are approximately 20 nm larger than THC-loaded liposomes. Liposomes were stored at 4 °C . Their stability has been monitored for 5 weeks. No signs of aggregations were observed during this time.

Cannabinoid encapsulation was assessed by HPLC, and encapsulation efficiencies of 50% and 60% were observed for THC- and CBD-loaded formulations, respectively (Fig. 5).

Figure 5. Encapsulation efficiency of cannabinoids in POPC liposomes.

The size differences between THC- and CBD-loaded liposomes coincide with the higher encapsulation efficiencies for the CBD-loaded liposomes. Given that cannabinoids are hydrophobic drugs, they are expected to partition into the lipophilic portion of the bilayer. CBD has less rigid structure than THC and there is a possibility that it could better fit into the intermolecular spaces in the bilayer. This could explain the higher EEs for CBD-loaded liposomes as well as more noticeable size increase. However, the bilayer space is still limited by the structure and ratios of the lipids constituting liposomes, so overall size change is relatively small. 



Liposomes containing POPC, cholesterol, and a DSPE-PEG2000 were formulated and in situ-loaded with cannabinoids using the NanoAssemblr Benchtop instrument. Liposome size was tuned using instrument process parameters. Stable THC- and CBD-loaded liposomes of  < 90 nm size were obtained. Cannabinoid encapsulation efficiencies of 50% for THC  and 60% for CBD were achieved. CBD-loaded liposomes have higher encapsulation efficiencies and larger size than THC-loaded ones. This suggests CBD may intercalate more effectively between lipid tails than more rigid THC, but further research is needed to understand these observations. 

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  2. Sharma, G. et al. Nanoparticle based insulin delivery system: the next generation efficient therapy for Type 1 diabetes. Journal of Nanobiotechnology 13, (2015).
  3. Ohlsson, A. et al. Plasma delta-9-tetrahydrocannabinol concentrations and clinical effects after oral and intravenous administration and smoking. Clinical Pharmacology and Therapeutics 28, 409–416 (1980).
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  7. Zhigaltsev, I. V. et al. Bottom-Up Design and Synthesis of Limit Size Lipid Nanoparticle Systems with Aqueous and Triglyceride Cores Using Millisecond Microfluidic Mixing. Langmuir 28, 3633–3640 (2012).
  8. Zhigaltsev, I. al. Production of limit size nanoliposomal systems with potential utility as ultra-small drug delivery agents. Journal of Liposome Research 1–7 (2015). doi:10.3109/08982104.2015.1025411
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Employing nanoparticle formulation technology from the cutting edge of genetic medicine, Ascension Sciences is developing cannabinoid nano delivery platforms and techniques for the pharma and nutraceutical industries. Liposomes, nanoemulsions, lipid nanoparticles and polymeric nanoparticles have all shown promise in improving the therapeutic benefits of cannabinoids, but the full potential of these therapies has not yet been unlocked.

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