CASE STUDIES
Nanobiotechnology formulation for the delivery of drugs / imaging agent
Client Profiles
A privately-held company is focussed on the development of novel therapeutic approaches based on stem cell therapy.
Business Objectives
The company needs to make nanospheres of dye loaded poly(lactic-co-glycolic acid) (PLGA) polymer for a pharmaceutical formulation.
Technical Challenges
Making biodegradable nanospheres is an art. The challenge is in the ability to retain the encapsulated drug and/or imaging agent at the nanometer level. To produce spherical nanoparticles with an intact active ingredient, the processing conditions for making nanoparticles require relatively high pressure, minimum shear and minimal solvent rates of evaporation.
Realization
Equipment:
| Microfluidizer processor: |
M-110Y |
| Microscope: |
Olympus BH-2 optical with attachments |
| Diluent: |
De-ionized water (17.1 MegaOhms) |
| Rotovap: |
Buchi Rotovapor R110 |
| Chamber Configuration: |
F20Y (75 µm) - H30Z (200 µm) |
| Particle Size Analyzer: |
Malvern Zetasizer |
| Rotostator Mixer: |
IKA T25 |
| Refractive Index: |
1.33 – 0.1i |
Procedure:
The PLGA and the dye were first dissolved in dichloromethane (DCM), then mixed with a mixture of water and poly(vinyl alcohol) (PVA) to form a coarse emulsion. The two streams were mixed together for one minute using a roto-stator mixer. The sample was then processed at 10,000 psi for one, two, and three passes with the F20Y (75 µm) chamber and the H30Z (200 µm) chamber. All samples were processed using the M-110Y Microfluidizer processor. After each discrete pass a portion of the sample was placed in a rotovap to remove the DCM.
Analysis:
Analysis involved the use of the optical microscope and a dynamic light scattering particle size analyzer (Malvern Zetasizer).
Results:
Table 1 list the processing conditions and the average particle size of the samples. Microscope images are shown in Figure 1.
Table 1: Particle size analysis after various stages of processing. All samples were processed with the M-110Y Microfluidizer processor and with the F20Y (75 µm) IXC and H30Z (200 µm) APM.
| Passes |
Pressure (psi) |
Mean (nm) |
| 1 Pass |
10,000 |
243,4 |
| 2 Passes |
10,000 |
176,8 |
| 3 Passes |
10,000 |
169,5 |
Figure 1: Optical images after various passes at 10,000 psi using the M-110Y Microfluidizer processor and the F20Y (75 µm) IXC and H30Z (200 µm) APM chambers.
Conclusion
Using optimal processing conditions we were successful in creating polymer PLGA nanospheres with an average particle size of 169.5 nm after 3 passes at 10,000 psi using the F20Y (75 µm) interaction chamber (IXC) and H30Z (200 µm) auxiliary processing module (APM).
Liposomal formulation development |
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Client Profiles
A privately-held biotech company founded in 2004 around a distinctive, proprietary, technology platform that uses synthetic biology to create innovative small molecule drugs and other compounds. In particular their platform allows the creation of chemistries that recapitulate the themes found in natural products, but without the disadvantages of those approaches.
Business Objectives
The Client was looking for a liposomal formulation designed to encapsulate their proprietary antifungal drug to reduce its intrinsic toxicity. The objective of this project was to produce liposomal formulations of client’s proprietary pharmaceutical ingredient and to determine the in vitro release rate.
Technical Challenges
The ability to encapsulate the API had never been done before. The proper exchange buffers for incorporating the API using an active method had to be developed. A high encapsulation efficiency had to be obtained with a corresponding in-vitro release rate correspong to the target product profile.
Realization
Formulation components:
The Client furnished the API and the lipid formulation excipients as shown in Table 1. The API is a yellowish substance and granular in appearance at room temperature and it is light-sensitive. About 5 grams of this API were provided by the client for this preliminary aspect of the work.
Table 1: Formulation Excipients
| Excipient |
Mole % |
| DSPE PEG 2000 |
5% |
| DMPC |
55% |
| Cholesterol NF |
40% |
All reagents used in this study were analytical reagent grade or better. Source of unique reagents will be noted after the first mention of each chemical within the text of the final report.
Methods and Procedures
The pegylated liposomes had a lipid composition as shown in Table 1. To form unilamellar vesicles, each of the liposomal formulations was extruded at least once using a mini extruder (Avanti Polar Lipids # 610000). The active liposomal API loading approach was adopted in order to enhance the API drug encapsulation. The internal buffer used was citrate buffer at pH 4 which would cause a precipitate of the drug to form in the interior of the liposomes. On the other hand, the API was soluble in 150 mM NaCL 25Mm HEPES buffer at pH 7.5. The drug loading ratio was 2 mg of API to 10 mg of lipids. The liposomal drug loading took 40 minutes while maintaining a temperature of 40 °C, which is above the lipid transition temperature for this formulation. The liposomal formulations were then buffer-exchanged using dialysis to remove unwanted buffer and unencapsulated API. The intermediate drug product was then transferred into 50 cc centrifuge tubes and lyophilized for 48 hours. The post-lyophilized API drug product was inspected and packaged for delivery.
Analytical Test to determine the encapsulation efficiency.
Isopropyl alcohol (IPA) was used to disrupt the liposomes and the API concentration was measured by HPLC using UV/Vis detection at 310 nm.
In-vitro test
The lyophilized drug product was reconstituted and tested for API drug release kinetics using Franz Cell diffusion cells equipped with a 0.45 µm synthetic membrane (Tuffryn™ - Pall® Corporation). This in vitro testing was performed continuously for 24 hours.
Results
The results are shown in Tables 2 and 3. For lots BV002-A, BV002-B and BV002-C, the percent drug recoveries obtained were 95%, 82% and 85% respectively as shown in Table 2.
The results of Franz Cell diffusion test are shown in Table 3. Lot BV002-003A showed a drug release of 3.4% within 24 hrs while BV002-003B and BV002-003C exhibited drug releases of 7.3% and 8.8% respectively, which reflects a stable encapsulation of the API. Greater release rates are expected in vivo due to liposomal entrapment by the target tissue (unpublished data).
Table 2: API Recovery from the liposomal formulation
| Lot # |
API Encapsulation (mg) |
API Recovery (%) |
| BV002-A |
17 |
95 |
| BV002-B |
16 |
82 |
| BV002-C |
17 |
85 |
Table 3: Franz Cell Release Test
| Lot # |
API Release after 25.5 hours or testing (%) |
| BV002-003A |
3.4 |
| BV002-003B |
7.3 |
| BV002-003C |
8.8 |
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