This article describes a process for tuning the size and crosslinking density of covalently crosslinked nanoparticles from linear polyesters containing pendant functionality. By tailoring synthesis parameters (polymer molecular weight, pendant functionality incorporation, and crosslinker equivalents), a desired nanoparticle size and crosslinking density can be achieved for drug delivery applications.
We describe a protocol for the synthesis of linear polyesters containing pendant epoxide functionality and their incorporation into a nanosponge with controlled dimensions. This approach begins with synthesis of a functionalized lactone which is key to the pendant functionalization of the resulting polymer. Valerolactone (VL) and allyl-valerolactone (AVL) are then copolymerized using ring-opening polymerization. Post-polymerization modification is then used to install an epoxide moiety on some or all of the pendant allyl groups. Epoxy-amine chemistry is employed to form nanoparticles in a dilute solution of both polymer and small molecule diamine crosslinker based on the desired nanosponge size and crosslinking density. Nanosponge sizes can be characterized by transmission electron microscopy (TEM) imaging to determine the dimension and distribution. This method provides a pathway by which highly tunable polyesters can create tunable nanoparticles, which can be used for small molecule drug encapsulation. Due to the nature of the backbone, these particles are hydrolytically and enzymatically degradable for a controlled release of a wide range of hydrophobic small molecules.
Precisely tuning the size and crosslinking density of nanoparticles based on intermolecular crosslinking is of great importance to influence and guide the drug release profile of these nanosystems1. Designing nanosponge tunability, i.e., preparing particles of different network densities, is reliant upon the pendant functionality of the precursor polymer and the equivalents of the hydrophilic crosslinker incorporated. In this approach, the concentration of the precursor and crosslinker in the solvent is important to form nanoparticles of a discrete size rather than a bulk gel. Utilizing quantitative nuclear magnetic resonance spectroscopy (NMR) as a characterization technique allows for the precise determination of incorporated pendant functionality and polymer molecular weight. Once nanoparticles are formed, they can be concentrated and solubilized in organics without having the character of a nanogel.
Recent work in nanoparticle drug delivery has focused on the use of poly(lactic-co-glycolic acid) (PLGA) self-assembled nanoparticles2,3,4,5,6. PLGA has degradable ester linkages which make it suitable for drug delivery applications and is often combined with poly(ethylene glycol) (PEG) due to its stealth properties7. However, due to the self-assembled nature of PLGA particle formation, the particles cannot be solubilized in organics for further functionalization. In contrast to PLGA nanoparticles, the proposed method provides covalent crosslinking forming a nanoparticle with defined sizes and morphology, which are stable in organics and degrade in aqueous solutions1. Advantages of this approach are the ability to further chemically functionalize the surface of the nanosponge8, and its stability in organic solvents can be used for the post-loading of the particles with pharmaceutical compounds1,9. With this method, encapsulation of hydrophobic small molecules can be achieved by precipitation into aqueous media. The hydrophobicity of the polyester backbone together with the hydrophilic short crosslinker gives these particles an amorphous character at body temperature. Furthermore, after drug loading, the particle can form fine suspensions in aqueous media to be readily injected in vivo. It is our goal in this work to evaluate the parameters for the synthesis of these polyester nanosponges and determine those which are vitally important to the design and control of size and morphology.
1. Synthesis and Characterization of AVL
2. Synthesis and Characterization of VL- co- AVL
3. Post-polymerization Epoxidation to Produce Epoxy-valerolactone (EVL) Copolymer Units
4. Nanosponge Synthesis and Characterization
5. TEM Imaging of Nanosponge Morphology and Size
To evaluate the relationship between the synthesis parameters of the nanosponge and its resultant size, the concentration and pendant functionality of each polymer precursor is important. In Figure 1, a successfulsynthetic scheme of nanosponges is carried out under reflux conditions after incorporating both precursor polymer and diamine crosslinker in DCM for 12 h. The concentration of epoxides in the solution is also critical to forming discrete particles. Once nanosponges were synthesized, TEM imaging was used to determine the precise dimensions of a set of particles. In Figure 2, a collection of various nanosponge experiments was analyzed based on their polymer precursor molecular weight and pendant functionality incorporation to determine if a relationship between the two could have an effect on the nanosponge size. In Figure 2, a trend of increasing particle size is seen as molecular weight is increased for both a 6% and 8% EVL incorporation with one diamine crosslinker per epoxide (2 amines per epoxide).
Figure 3 shows that increasing both the epoxide percentage and crosslinker equivalents would have a similar effect while maintaining a similar molecular weight between nanosponge sets. Again, a trend in increasing nanosponge size while changing these parameters is seen. It is important to understand how the synthesis of polymer precursors can play a vital role in the resultant nanoparticle size to precisely tune nanosponges for various applications. It is also important to maintain a reproducible and reliable method for nanosponge synthesis which has small deviations among individual particle size, as shown by Figure 4. By utilizing these parameters, a range of sizes and a formula for reliably reproducing a nanosponge of a particular size can be developed for a given application or desired goal, proving this to be a versatile and practical nanosponge chemistry.
Figure 1: Reaction Scheme for Nanosponge Synthesis. A linear polyester copolymer containing pendant allyl and epoxide functional groups is reacted with a diamine crosslinker to form discrete nanoparticles with size dimensions of approximately 100 nm. Please click here to view a larger version of this figure.
Figure 2: Analysis of Nanosponge Tunability Based on Molecular Weight and Pendant Functionality. By evaluating the change in the nanosponge size based on the molecular weight of the precursor polymer while keeping the relative pendant functionality the same, an increase in the particle size as the molecular weight increases can be shown for both 6% and 8% EVL polymers. Please click here to view a larger version of this figure.
Figure 3: Analysis of Nanosponge Tunability by Crosslinker and Pendant Functionality Equivalencies. By holding the crosslinker equivalent steady, a higher pendant functionality will result in higher crosslinker incorporation. In this figure, four amines per epoxide (two diamine crosslinker equivalents per epoxide) were added to both a 6% and 10 % EVL polymer. As more crosslinker is incorporated into the nanosponge due to more epoxides per polymer and higher crosslinker equivalents, the size increases. Please click here to view a larger version of this figure.
Figure 4: TEM Image of Nanosponges. A TEM image of covalently linked nanoparticles formed during synthesis. Indicated size of 79 ± 12 nm. Please click here to view a larger version of this figure.
Obtaining reproducible nanosponge sizes is vital in drug delivery applications. Multiple parameters in polymerization and nanosponge synthesis affect the size and crosslink density of the resulting particle. Three important parameters were identified in our analysis: polymer molecular weight, epoxide pendant functionality, and crosslinker equivalents. In order to produce a range of molecular weights and epoxide functionalities for nanosponge synthesis, the stoichiometry of the VL-co-AVL copolymer must be altered. The concentration of the allyl functional group during epoxidation of the copolymer can be used to epoxidize either a desired percentage of allyls or all of them. If an excess of oxidizing agent is used, degradation of the polymer chain can occur; however, this can be remedied by reducing the amount of oxidizing agent. When all allyls are epoxidized, there are no pendant allyls on the surface of the nanosponge for further functionalization. It is also important for nanosponge synthesis that the concentration of epoxide in solution nanosponge synthesis is 0.0054 M.
The nanosponge reaction has been previously evaluated to determine an optimal concentration for desired nanosponge size ranges13. This concentration is calculated based on the repeat unit value for the epoxide functionality in the polymer. The repeat unit is the weight of polymer per one reactive unit, which is used to calculate the moles of reactive units in one polymer. For example, as shown below, if a polymer with a molecular weight of 2,000 g/mol contains 10 reactive monomer units (RMU) bearing pendant functionality, determined by quantitative NMR, the reactive unit of the polymer is 200 g/mol RMU. Using this value, the moles of reactive units can be calculated from the polymer weight in order to determine crosslinker equivalencies for nanosponge synthesis.
As a general trend, increasing both the polymer molecular weight and epoxide functionality contributed to an increased nanosponge size independently. A narrow polydispersity achieves a narrow nanosponge size distribution (~ 10% standard deviation) and improves reproducibility of nanosponge synthesis.
The presented approach achieves a narrow polymer dispersity by use of a tin triflate catalyst14. The crosslinking equivalencies are calculated based on the amine per epoxide equivalents, and an increase in crosslinker equivalents is shown to increase the nanosponge size. However, using an excess of crosslinker is important due to the goal of consuming all available epoxides. Remaining amine functionality on the nanosponge surface can be used for further functionalization of the particle surface.
Compared to conventional methods for nanoparticle preparation, the advantages in this approach are the multiple parameters by which precise size and density control can be achieved, the ability to further functionalize the surface of the nanosponge, and the solubility in the organics for hydrophobic drug encapsulation.
The authors have nothing to disclose.
LK is thankful for funding from the National Science Foundation Graduate Research Fellowship Program (DGE-1445197) and Vanderbilt University Chemistry Department. LK and EH would like to thank the funding for the Osiris TEM instrument (NSF EPS 1004083).
2,2'-(Ethylenedioxy)bis(ethylamine) | Sigma-Aldrich | 385506-100ML | |
3-methyl-1-butanol | Sigma-Aldrich | 309435-100ML | anhydrous, ≥99% |
Acetone | Sigma-Aldrich | 179124-4L | |
Allyl bromide | Sigma-Aldrich | A29585-5G | ≥99% |
Ammonium chloride | Fisher Scientific | A661-500 | saturated solution in DI water |
Cell culture water | Sigma-Aldrich | W3500-500ML | Filtered through 0.45 μm syringe filter |
Dichloromethane (DCM) | Sigma-Aldrich | 270997-100ML | anhydrous, ≥99%, contains 40-150 ppm amylene as stabilizer |
Ethyl Acetate | Fisher Scientific | E145SK-4 | |
EZFlow 0.2 μm Syringe Filter | Foxx Life Sciences | 386-2116-OEM | Hydrophillic PTFE, 13 mm |
EZFlow 0.45 μm Syringe Filter | Foxx Life Sciences | 386-3126-OEM | Hydrophillic PTFE, 25 mm |
Fisherbrand Disposable Borosilicate Glass Test Tubes with Plain End | Fisher Scientific | 14-961-31 | |
Fisherbrand Microcentrifuge Tubes | Fisher Scientific | 14-666-318 | 1.5 mL |
Hamilton Microliter Syringe, 100 μL | Hamilton Company | 80600 | Model 710 N SYR, Cemented NDL, 22s ga, 2 in, point style 2 |
Hexamethylphosphoramide | Sigma-Aldrich | H11602-100G | ≥99%, contains ≤1000 ppm propylene oxide as stabilizer |
Hexanes | Fisher Scientific | H292-4 | |
Magnesium sulfate anhydrous | Fisher Scientific | M65-500 | |
Meta-chloroperoxybenzoic acid | Sigma-Aldrich | 273031-100G | Purified to ≥99% by buffer wash |
Methanol (MeOH) | Sigma-Aldrich | 322415-100ML | anhydrous, ≥99% |
N-butyllithium solution | Sigma-Aldrich | 230707-100ML | 2.5 M in hexanes |
N,N-diisopropylethylamine | Sigma-Aldrich | 550043-500ML | ≥99% |
Parafilm M | Sigma-Aldrich | P7793-1EA | |
PELCO Pro Reverse (Self-Closing) Tweezers | Ted Pella, Inc. | 5375-NM | |
Phosphotungstic acid hydrate | Alfa Aesar | 40116 | |
Q55 Sonicator | Qsonica | Q55-110 | 55 Watts, 20 kHz |
SiliaMetS Cysteine | Silicycle | R80530B-10g | |
SnakeSkin Dialysis Clips | Thermo Scientific | 68011 | |
SnakeSkin Dialysis Tubing, 10K MWCO | Thermo Scientific | 68100 | |
Sodium bicarbonate | Fisher Scientific | 5233-500 | saturated solution in DI water |
TEM grid | Ted Pella, Inc. | 01822-F | Ultrathin Carbon Type-A, 400 mesh, Copper, approx. grid hole size: 42µm |
Tetrahydrofuran (THF) | Sigma-Aldrich | 401757-1L | Anhydrous, ≥99.9%, inhibitor-free |
Tin(II) trifluoromethanesulfonate | Sigma-Aldrich | 388122-1G | |
Vortex-Genie 2 | Scientific Industries | SI-0236 | |
Whatman Filter Paper, Grade 1 | Fisher Scientific | 09-805H | Circles, 185 mm |
δ-valerolactone | Sigma-Aldrich | 389579-100ML | Purified by vacuum distillation |