Highly scalable, closed-loop synthesis of drug-loaded, layer-by-layer nanoparticles

Abstract

Layer-by-layer (LbL) self-assembly is a versatile technique from which multicomponent and stimuli-responsive nanoscale drug carriers can be constructed. Despite the benefits of LbL assembly, the conventional synthetic approach for fabricating LbL nanoparticles requires numerous purification steps that limit scale, yield, efficiency, and potential for clinical translation. In this report, we describe a generalizable method for increasing throughput with LbL assembly by using highly scalable, closed-loop diafiltration to manage intermediate purification steps. This method facilitates highly controlled fabrication of diverse nanoscale LbL formulations smaller than 150 nm composed from solid-polymer, mesoporous silica, and liposomal vesicles. The technique allows for the deposition of a broad range of polyelectrolytes that included native polysaccharides, linear polypeptides, and synthetic polymers. We also explore the cytotoxicity, shelf life and long-term storage of LbL nanoparticles produced using this approach. We find that LbL coated systems can be reliably and rapidly produced: specifically, LbL-modified liposomes could be lyophilized, stored at room temperature, and reconstituted without compromising drug encapsulation or particle stability, thereby facilitating large scale applications. Overall, this report describes an accessible approach that significantly improves the throughput of nanoscale LbL drug-carriers that show low toxicity and are amenable to clinically relevant storage conditions.

Keywords: biomaterials; colloid chemistry; layer-by-layer nanoparticles; polymer engineering; scalable synthesis.

Figure 1

Tangential flow filtration (TFF) facilitates the rapid and controlled fabrication of layer-by-layer nanoparticles. (a) TFF purification schematic depicting continuous diafiltration through a porous membrane. A peristaltic pump drives nanoparticle samples through a circuit containing a filter membrane. Polyelectrolytes, driven by a mild pressure gradient, exit the circuit through the pores in the filter membrane and into a waste reservoir. Removal of solution generates a vacuum within the system, which draws replacement buffer from an attached reservoir to hold sample volume constant. To prevent nonspecific adsorption onto the filter membrane, purification of cationic and anionic nanoparticles was separated to different purification loops, each with its own filtration membrane. The left panel denotes the cationic purification loop, and the right panel denotes the anionic purification loop. (b) Purification of excess poly-L-lysine (PLL) or dextran sulfate (DXS) following LbL deposition can be completed in minutes using TFF. The concentration of PLL extracted using TFF was determined using a BCA assay on samples taken sequentially from the waste stream. DXS concentrations were determined by analyzing permeate samples by gel permeation chromatography. Data were fitted using a one-phase exponential decay model. (c) High yields (68 ± 5% after 10 layers) are reproducibly obtained using TFF-assisted LbL fabrication. Nanoparticle concentration was quantified using a fluorescence plate reader following each purification step during the fabrication of 100 nm, carboxy-modified latex particles coated with five bilayers of PLL and DXS (CML-[PLL/DXS]₅). Error bars represent standard deviation of three independent syntheses.

Figure 2

Diverse layer-by-layer (LbL) nanoparticles are prepared in a controlled manner using the tangential flow filtration (TFF)-assisted method. (a–c) Solid-core LbL particles were prepared by coating 100 nm, carboxy-modified latex particles with five bilayers of poly(L-lysine) and dextran sulfate (100CML-[PLL/DXS]₅). (d–f) Liposomal-core LbL particles were prepared by coating negatively charged, doxorubicin-containing liposomes with four bilayers of PLL and DXS followed by a bilayer of PLL and heparin sulfate (Lipo-[PLL/DXS]₄-PLL-HS). (a, d) These particles exhibited controlled size increase during layer deposition. (b, e) Nanoparticle uniformity was maintained throughout the layering process as indicated by the low polydispersity index (PDI). (c, f) Complete charge reversal was observed after each layer deposition indicating successful LbL modification. Size and polydispersity data were acquired by dynamic light scattering, and zeta potential data was measured using laser Doppler electrophoresis. Error bars represent standard deviation of three technical replicates. For population-based data see Supplemental Figure S5.

Figure 3

Cryogenic TEM images of core particles before and after layering confirm the presence of a thin film. Cryogenic TEM of (a) uncoated, carboxy-modified latex particles; (b) purified, LbL-coated carboxy-modified latex (CML-[PLL/DXS]₅); (c) uncoated, doxorubicin-loaded liposome; (d) purified, LbL-coated, doxorubicin-loaded liposome (Lipo-[PLL/DXS]₄-PLL-HS).

Figure 4

The quantification of Cryo-TEM images confirm statistically significant increases in size consistent with LbL modification. (a) Comparison of the diameters of bare, carboxy-modified latex (CML) particles and LbL-modified CMLs indicate a statistically significant (P<0.0001) shift in the size distribution. Dashed line denotes the mean size. N = 103 for bare and 153 for LbL CML. (b) Similarly, comparison of the liposome membrane thickness reveals statistically significant (P<0.0001) increase in thickness after LbL modification. Dashed lines denote the mean thickness. N = 113 for bare and 298 for LbL liposomes. (c) Calculation of LbL film thickness from either Cryo-TEM data or dynamic light scattering data give statistically consistent results for both CML (13.3 ± 0.6 nm by Cryo-TEM and 12 ± 1 nm by DLS) and liposomal (2.65 ± 0.09 nm by Cryo-TEM and 2 ± 1 nm by DLS) substrates. Error bars represent SEM. These data highlight important changes in LbL film thickness due to substrate selection. Individual particle measurements were plotted as a histogram and the kernel density function was estimated using R Statistical Software. All statistical tests were performed using one-way ANOVA (alpha = 0.01), with the Bonferroni post-test, on PRISM graphing software.

Figure 5

LbL nanoparticles prepared using the TFF-assisted method do not exhibit nonspecific cytotoxic effects in vitro. SKOV3 cells were incubated with poly(L-aspartic acid), herparin sulfate, and hyaluronic acid terminated LbL nanoparticles that were prepared by either the TFF method (solid, blue bars) or the conventional centrifugal method (striped bars). After 72 hours, the cellular viability was determined using the Cell-Titer Glo luminescence assay. The results were normalized relative to untreated controls, and subsequent analysis by one-way ANOVA failed to find any statistically significant changes in cellular viability for any formulation, regardless of preparation method.

Figure 6

LbL nanoparticles have long refrigerated shelf lives as indicated by the preservation of their size, uniformity and charge characteristics. Ten different LbL particles consisting of a carboxy-modified latex core and a bilayer of poly(L-arginine) and a unique polyanion were stored for three months at 4 degrees Celsius. (a) We compared the hydrodynamic number average size for the particles after storage and compared them to the particle’s original size. With the exception of the Fucoidan-coated particle, all others maintained statistically consistent sizes during storage. (b) These particles likewise exhibited statistically consistent polydispersity index, suggesting that particle uniformity is maintained during storage. (c) The z-average size measurement, which is more sensitive to aggregates, demonstrated that 7 out 10 formulations exhibited statistically consistent sizes during storage. (d) Generally, the zeta potential of the nanoparticles was conserved during storage with a few exceptions. Sulfated beta cyclodextrin-coated particles exhibited a significant charge increase during storage. On the other hand, hyaluronic acid and dextran sulfate-coated particles exhibited a small, but statistically significant decrease in zeta potential. Overall these data indicate that LbL particles would be amenable to months-long refrigerated storage without concern of loss of colloidal stability. One-way ANOVA with the Bonferroni post-test (alpha = 0.01) was used to determine statistical significance between the indicated samples.

Figure 7

Liposomal-core layer-by-layer nanoparticles can be lyophilized and reconstituted for long-term storage. Doxorubicin-loaded liposomes were coated with two bilayers of poly(L-arginine) and dextran sulfate (Lipo-[PLA/DXS]₂), using the TFF-method. (a) Lipo-[PLA/DXS]₂ particles were lyophilized with different cryopreservatives. (b) TEM of particles before lyophilization (left panel) and after being reconstituted from freeze-dried powder (right panel). (c) LbL modified liposomes retained more drug than bare liposomes regardless of choice of cryoprotectant, though several protectants facilitate nearly 100% drug retention during storage and reconstitution. (d) The presence of cryoprotectants prevents aggregation upon LbL liposome reconstitution. (e) Reconstituted LbL liposomes exhibit lower polydispersity index than reconstituted bare liposomes, indicating improved uniformity. (f) Cryopreservation with 10% sucrose or 11% trehalose prevented decreases in the zeta potential of LbL liposomes. Size and polydispersity data were acquired by dynamic light scattering, and zeta potential data was measured using laser Doppler electrophoresis. Error bars represent standard deviation of three technical replicates.