Effects of Concentrating Cannabinoid Loaded Liposome Formulations

April 2021 | By Dr. Natalia Sannikova, Research & Development Manager, and Andrea Olaizola, Formulation Associate Scientist at Ascension Sciences


Phytocannabinoids are chemical compounds found in the cannabis plant which have been increasingly used as  therapeutics in recent years [1]. They have shown to aid in chronic pain management for several diseases such as rheumatoid arthritis, ameliorate the side effects of chemotherapy, and relieve inflammation among other therapeutic benefits. 

However, cannabinoids possess low oral bioavailability given their hydrophobic nature [2]. Thus, special formulations are needed to ensure these bioactive compounds get absorbed by the digestive system to produce the desired effect. An effective way to increase the bioavailability of cannabinoids is by encapsulating them into nanoparticles [3]. Lipid nanoparticles are effective vehicles for hydrophobic bioactive compounds [4] and show strong potential for cannabinoid delivery. They are composed of a phospholipid bilayer and a hydrophilic core as shown in Figure 1. 

Figure 1. Liposome structure

This experiment aimed to compare the stability of cannabinoid-loaded liposomes formulated with synthetic and natural  phospholipids. POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) was chosen as the synthetic lipid and SoyPC (phosphatidylcholine isolated from the soybeans) as the natural. POPC contains one saturated 16-carbon acyl chain and one mono-unsaturated 18-carbon acyl chain. The main component of the SoyPC, 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLPC), consisted of one saturated 16-carbon acyl chain and one 18-carbon acyl chain with two double bonds (Fig. 2). 

Figure 2. Chemical structures of A) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and B) 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLPC).

Additionally, we assessed the effect of concentration on liposome stability. Formulations were concentrated by centrifugal filtration, and their stability was monitored for  35 days at 4°C. 

Materials and Methods

SoyPC was purchased from Avanti® Polar Lipids, DSPE-PEG2000 from Avanti® Polar Lipids, POPC and cholesterol were purchased from Sigma Aldrich. Extract from biomass of a high Δ9-THC chemotype of C. sativa was used for the experiments.

Corresponding phospholipid, cholesterol and PEGylated lipid were dissolved in absolute ethanol at proprietary ratios. 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 to form unilamellar liposomes. THC was loaded in situ by dissolving it in the lipid solution. Formulations were then dialyzed against PBS for ethanol removal

An aliquot of each formulation was concentrated after dialysis to approximately 10X using Amicon® Ultra-4 Centrifugal Filter Unit (purchased from Sigma Aldrich).

The stability of the liposomes was assessed by measuring the size and PDI (polydispersity index) using a Dynamic Light Scattering (DLS) system (Zetasizer, Malvern Instruments, UK). Samples were stored at 4°C and analyzed once a week for five weeks. Size and polydispersity index (PDI) were represented as the mean of three samples, error bars representing standard deviation of the mean. The liposome encapsulation efficiency (EE%) was determined by HPLC (high-performance liquid chromatography).

Lipid composition 

POPC : Chol : DSPE-PEG2000

SoyPC : Chol : DSPE-PEG2000

Organic solvent


Aqueous solvent

PBS pH 7.4

Solvent Removal


Lipid : API

10 : 1

Results and Discussion

Nanoparticle Stability

Liposomes with the different phospholipids were prepared with identical instrument parameters and API loads. The cholesterol amount was higher in the POPC-based formulation than in the SoyPC-based one, following what we have found to be the most optimal parameters for the two phospholipids. 

First, the unconcentrated liposome formulations were compared. The SoyPC-based liposomes showed slightly less variation in size and PDI over the 35 days of storage than the POPC-based liposomes (Figure 3). The POPC formulation particle size decreased after dialysis (Day 1) and then gradually increased. The PDI also decreased after dialysis, increased over the next two weeks of storage and then stabilized at 0.15 ± 0.01. Overall, both formulations demonstrated stability over 5 weeks of storage at 4°C. SoyPC-based liposomes had slightly higher particle size and PDI compared to the POPC-based, however not exceeding 100 nm in size and 0.25 in PDI, which characterizes both formulations as uniform with narrow size distribution and makes them promising candidates for cannabinoid delivery. 

Figure 3. POPC-based vs. SoyPC-based liposome stability up to 35 days post formulation.

Figure 4. Centrifugal filtration effect on liposome size and stability.

If desired, liposome concentration can be adjusted by dilution or by concentration using centrifugal filtration following NanoAssemblr formulation. Figure 3 demonstrates that liposomes can be concentrated to a final concentration as required with minimal effect on liposome size and PDI. The concentrated POPC liposome formulations showed to have a slightly smaller size than the original sample, however, the difference is not significant (Figure 4). For the unconcentrated POPC sample the size ranged from 73.2 nm to 87.6 nm and for the concentrated samples ranged from 77.0 nm to 87.6 nm. SoyPC-based liposomes have demonstrated similar behavior and can be concentrated after NanoAssemblr  Benchtop production by centrifugal filtration without appreciably affecting size.

Encapsulation Efficiency and Cannabinoid Concentration

Liposome encapsulation efficiency (EE%) was determined using the pre and post formulation THC concentration measured  by HPLC.

The [API]f is cannabinoid concentration post formulation and [API]d is cannabinoid concentration after the removal of non-encapsulated THC. The encapsulation efficiencies for POPC and SoyPC-based liposomes were 67% and 91%, respectively.

HPLC analysis of the supernatant after centrifugal filtration of the samples showed no traces of THC which proves centrifugation to be non-disruptive and suitable for concentrating liposomes. Corresponding concentration  coefficients of 12.2 for SoyPC-based  and 4.5 for POPC-based liposomes were determined based on the HPLC data. 

Figure 5. THC concentration before and after centrifugal filtration of formulations.


Stable liposome formulations for cannabinoid encapsulation can be prepared using  different unsaturated phospholipids as the main excipients. We have successfully produced stable formulations with comparable particle size and PDI and high encapsulation efficiency based on natural phosphatidylcholine from soybeans (SoyPC) and a synthetic lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine  (POPC).

Liposomes can be concentrated after NanoAssemblr Benchtop production by centrifugal filtration without appreciably affecting their size and stability.

  1. Whiting, P. F. et al. Cannabinoids for medical use: A systematic review and meta-analysis. JAMA – Journal of the American Medical Association vol. 313 2456–2473 (2015).
  2. Romero-Sandoval, E. A., Kolano, A. L. & Alvarado-Vázquez, & P. A. Cannabis and Cannabinoids for Chronic Pain. (1926) doi:10.1007/s11926-017-0693-1.
  3. Bruni, N. et al. Cannabinoid delivery systems for pain and inflammation treatment. Molecules vol. 23 (2018).
  4.  Daraee, H., Etemadi, A. & Kouhi, M. Application of liposomes in medicine and drug delivery. Samira Alimirzalu & Abolfazl Akbarzadeh 44, 381–391 (2016).

About Ascension Sciences

Employing nanoparticle formulation technology from the cutting edge of genetic medicine, Ascension Sciences Inc. (ASI) is developing cannabinoid nano delivery platforms and techniques for the pharma and nutraceutical industries. ASI’s R&D and formulation development services are an efficient option for research driven firms that require the advantages of nanoparticle delivery for their active ingredients.

 For more information, please visit www.ascensionsciences.com.