1Laboratory of Nano- and Translational Medicine, Department of Radiation Oncology, Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, 2Carolina Center for Nanotechnology Excellence, University of North Carolina
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Karve, S., Werner, M. E., Cummings, N. D., Sukumar, R., Wang, E. C., Zhang, Y., et al. Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique. J. Vis. Exp. (55), e3398, doi:10.3791/3398 (2011).
Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale (nanoparticles). Nanoparticles can be engineered in a precise fashion where their size, composition and surface chemistry can be carefully controlled. This enables unprecedented freedom to modify some of the fundamental properties of their cargo, such as solubility, diffusivity, biodistribution, release characteristics and immunogenicity. Since their inception, nanoparticles have been utilized in many areas of science and medicine, including drug delivery, imaging, and cell biology1-4. However, it has not been fully utilized outside of "nanotechnology laboratories" due to perceived technical barrier. In this article, we describe a simple method to synthesize a polymer based nanoparticle platform that has a wide range of potential applications.
The first step is to synthesize a diblock co-polymer that has both a hydrophobic domain and hydrophilic domain. Using PLGA and PEG as model polymers, we described a conjugation reaction using EDC/NHS chemistry5 (Fig 1). We also discuss the polymer purification process. The synthesized diblock co-polymer can self-assemble into nanoparticles in the nanoprecipitation process through hydrophobic-hydrophilic interactions.
The described polymer nanoparticle is very versatile. The hydrophobic core of the nanoparticle can be utilized to carry poorly soluble drugs for drug delivery experiments6. Furthermore, the nanoparticles can overcome the problem of toxic solvents for poorly soluble molecular biology reagents, such as wortmannin, which requires a solvent like DMSO. However, DMSO can be toxic to cells and interfere with the experiment. These poorly soluble drugs and reagents can be effectively delivered using polymer nanoparticles with minimal toxicity. Polymer nanoparticles can also be loaded with fluorescent dye and utilized for intracellular trafficking studies. Lastly, these polymer nanoparticles can be conjugated to targeting ligands through surface PEG. Such targeted nanoparticles can be utilized to label specific epitopes on or in cells7-10.
1. Synthesis of PLGA-b-PEG polymer
2. PLGA-b-PEG nanoparticle preparation
Nanoparticles with PLGA core covered with PEG at the surface can be prepared with these diblock copolymers. A variety of different hydrophobic drugs can be encapsulated in such nanoparticles. Fluorescent compounds can be encapsulated in the nanoparticles or can be conjugated to PLGA and thus these nanoparticles can be used for fluorescence imaging.
Nanoprecipitation method is used to make nanoparticles especially when the desired cargo to be encapsulated is highly hydrophobic in nature.
Freeze-drying is a commonly used method to store nanoparticles11. Freeze-drying will preserve the physical and chemical characteristics of the nanoparticles for long term stability12. The freeze drying process can cause stress on the particles and destabilize the formulation, so cryo-protectants (protection from freezing stress) and lyo-protectants (protection from drying stress) are commonly used. The choice of these protectants is determined by the desired length of storage time13.
4. Representative results:
Characterization of PLGA-b-PEG Di-block Copolymer
Different techniques can be used to confirm the successful conjugation of polymers. The composition of PLGA-b-PEG can be characterized using a 400 MHz 1H nuclear magnetic resonance (NMR). Molecular weight of the formed product (PLGA-b-PEG) can be verified by Gel permeation chromatography (GPC). The PLGA-b- PEG molecular weight distribution curve and elution time should be different from PLGA and PEG alone. In combination, these techniques should characterize the formed product and determine whether the conjugation reaction was successful.
Characterization of PLGA-b-PEG nanoparticles
Particle size and size distribution can be measured by dynamic light scattering. Different parameters in the nanoprecipitation process affect the size of particles. Molecular weights of the polymers used initially (both PLGA and PEG) also effect the particle size distribution. Transition electron microscopy (TEM) can also be used to confirm the size distribution and structure of the nanoparticles as seen in figure 3. The particle size range is generally in the nm range. Large particle sizes with uneven size distribution could indicate either an error in the conjugation reaction or the nanoprecipitation method needs optimization. In addition, surface zeta potential can be measured by ZetaPALS.
The drug/cargo loading efficiency can be quantified with standard HPLC.
The particles are dissolved in organic solvent and HPLC can be performed to measure the absorbance of the drug/cargo (Fig. 4). The drug release kinetic study can be done where known fixed quantities of the nanoparticles are dialyzed in 30 Slide-A-Lyzer MINI Dialysis units. At fixed time intervals, the content in the dialysis unit is collected and equal volume of organic solvent is added to dissolve the nanoparticles. HPLC is done on these samples to quantify the drug/cargo content.
Figure 1. EDC/NHS chemistry
Figure 2. Nanoprecipitation method for preparing polymeric nanoparticles. The organic solution of a solvent (acetonitrile or DCM) containing the PEG-PLGA diblock and the drug or cargo to be loaded into the particle is added dropwise to 3-5 mL of stirring H2O.
Figure 3. Transmission Electron Microscopy of nanopartices. A TEM image of PEG-PLGA nanoparticles containing wortamin. Phosphotungstic acid was used as a contrast agent.
Figure 4. Controlled release of drug from nanoparticle. Release of Paclitaxel from nanoparticles after dialysis in PBS. At the noted time, particles were removed from dialysis cassettes and solublized in acetonitrile. The solution was measured by HPLC. Two separate lots of nanoparticles were compared.
The nanoprecipitation method using diblock co-polymers represents a simple, fast method to engineer polymeric nanoparticles. The resulting nanoparticles are composed of a hydrophobic core which can be utilized for the delivery of poorly soluble compounds. The surface hydrophilic layer enables excellent aqueous solubility while providing a moiety for potential further conjugation to a targeting ligand.
There are many nanoparticle platforms, including liposomes, polymeric nanoparticles, dendrimers, metal particles, and quantum dots14. Among these platforms, the polymeric nanoparticle platform is one of the easiest to formulate and the most versatile in terms of applications. It requires minimal equipment setup and the technique can be learned in several hours. It also has a wide range of applications and its biocompatibility enables both in vitro and in vivo applications. Its ability to carry a cargo allows imaging and therapeutic capabilities.
EDC/NHS chemistry is presented here to generate the diblock copolymer. However, block copolymers can be synthesized using different catalysts. Another commonly used catalyst is stannous octoate. The terminal hydroxyl groups of PEG are used as initiating groups to synthesize block copolymers. Ring polymerization of lactide and glycolide initiated by dihydroxy PEG or monomethoxy PEG can lead to A-B-A or A-B type block copolymers respectively15. This method of preparation gives more flexibility in the design, however the EDC/NHS chemistry is easier to use and can save time by using a commercially available PLGA polymer.
In addition to nanoprecipitation, other methods to generate diblock polymer nanoparticles can be used. A common alternative is an "oil in water" emulsion method16. The emulsion method again starts with an organic phase containing the diblock copolymer and an aqueous phase. However, upon mixing the two solutions, nanoparticles are generated through vortexing and sonicating. This method is very similar, but the nanoprecipitaion method allows more control in the mixing step as well as avoids the use of sonication.
There are many potential applications for this platform. First, it can be utilized for the delivery of hydrophobic/poorly soluble drugs in drug delivery studies. For example, taxanes are poorly soluble and require a solvent for in vivo studies. Polymeric nanoparticles can encapsulate taxane drugs and abrogate the need for solvents. Nanoparticles can also deliver cell biology reagents that are poorly soluble, such as wortmannin. Polymer nanoparticles can also be loaded with fluorescent dye and utilized for intracellular trafficking studies. These polymer nanoparticles can be conjugated to targeting ligands through surface PEG. Combined with fluorescent labeling, these targeted nanoparticles can be used to label specific epitopes on or in cells. Since each nanoparticle can encapsulate a large number of fluorescent molecules, the nanoparticles can improve the sensitivity of such biological studies. Fluorescent labeled nanoparticles can also be utilized for in vivo imaging, such as the visualization of blood vessels and atherosclerotic plaques.
No conflicts of interest declared.
This work was funded by the Golfers Against Cancer, Carolina Center for Nanotechnology Excellence Pilot grant, University Cancer Research Fund and National Health Institute K-12 Career Development Award.
|EDC||Thermo Fisher Scientific, Inc.||22980||Conjugation Reagent|
|NHS||Thermo Fisher Scientific, Inc.||24500||Conjugation Reagent|
|amine-PEG-carboxylate||Laysan Bio Inc.||Nh2-PEG-CM-5000||Polymer (Can use any PEG MW, 5000 is listed here)|
|Acetonitrile >99% purity||Sigma-Aldrich||34851||Solvent|
|Methanol >99% purity||Sigma-Aldrich||34860||Wash|