NOTE: The animal studies included in all procedures have been approved by the Institutional Animal Care and Use Committee at the University of North Texas Health Science Center.
1. Preparation of NGF HDL-mimicking Nanoparticles
2. Characterization of NGF HDL-mimicking Nanoparticles
3. In Vitro Release of NGF HDL-mimicking Nanoparticles
4. Bioactivity of NGF HDL-mimicking Nanoparticles (Neurite Outgrowth Study)
5. Biodistribution of NGF HDL-mimicking Nanoparticles
The engineering scheme of HDL-mimicking, α-tocopherol-coated NGF NPs prepared by an ion-pair strategy is shown in Figure 1. To neutralize the surface charges of NGF, protamine USP was used as an ion-pair agent to form a complex with NGF. To protect the bioactivity, prototype HDL-mimicking NPs were engineered, first using homogenization; then, the NGF/protamine complex was encapsulated into the prototype NPs. Homogenization provided sufficient energy and successfully promoted the mixing of the excipients. After a 3 min homogenization, consistent particle sizes (around 170 nm) were obtained for the prototype NPs (Figure 2). Apo A-I was incubated with the prototype NPs in different conditions, including 2 h of stirring at room temperature; 4 h of stirring at room temperature; 4 h of stirring at room temperature, followed by overnight incubation at 4 °C; and stirring at room temperature overnight. Over 26% of Apo A-I was incorporated in the NPs when stirred at room temperature overnight. To add the NGF-protamine complex, the complex was incubated with the prototype NPs for 30 min at 37 °C and then Apo A-I was added to the mixture to finish the final coating of Apo A-I on the surface of the NPs. Using the procedure described here, the final NGF HDL-mimicking NPs had particle sizes of 171.4 ±6.6 nm (n = 3), with 65.9% of NGF entrapment efficiency (Table 1). NGF HDL-mimicking NPs had a slight negative charge (Table 1). The NPs had a narrow size distribution, and incorporating NGF into the NPs did not affect the particle size (Figure 3).
To measure the entrapment efficiency of NGF, various methods were evaluated to separate unloaded NGF from NGF HLD-mimicking NPs. Unexpectedly, NGF cannot pass a separation filter (molecular weight cutoff: 100 kDa). Gel filtration columns, including Sephadex G-50, Sephadex G-100, and Sephacryl S-100, cannot separate unloaded NGF and NGF HDL-mimicking NPs, since both of them come out in the same fractions after elution. A Sepharose CL-4B column performed the separation with the optimized sample loading, elution buffer, and elution rate. As shown in Figure 4, unloaded NGF and NGF HDL-mimicking NPs were completely separated on a Sepharose CL-4B column.
A dialysis method was used to study the in vitro release of NGF HDL-mimicking NPs in PBS with 5% BSA, which was included to mimic the physiological conditions in blood. By increasing the size of the dialysis device (molecular weight cutoff: 300 kDa) and by adding PBS and BSA to the release medium, NGF did not bind with the dialysis membrane and freely passed through. As a result, the recovery of free NGF in this dialysis method was over 85% (Figure 5). NGF HDL-mimicking NPs showed a slow release profile, and about 10% of the NGF was released from the NPs over 72 h (Figure 5).
To test the bioactivity of NGF HDL-mimicking NPs, the subculture of PC12 cells that had a strong adhesion property was selected to conduct a neurite outgrowth assay. Figure 6 represents the imaging of neurite outgrowth when the cells were treated with 50 ng/mL free NGF (Figure 6A) and NGF HDL-mimicking NPs (Figure 6B). When the treatment concentration was higher than 10 ng/mL, neurite outgrowth was clearly observed by the microscope. At these high concentrations, free NGF and NGF HDL-mimicking NPs did not show a significant difference on the effect of neurite outgrowth. When the concentration of NGF was lower than 10 ng/mL, neurite outgrowth could not be observed clearly, both for free NGF and for NGF HDL-mimicking NPs. Biodistribution studies were performed to compare the in vivo behaviors of free NGF and NGF HDL-mimicking NPs. As shown in Figure 7, NGF HDL-mimicking NPs significantly increased the plasma concentration and decreased the uptake in the liver, kidney, and spleen.
Figure 1: The engineering scheme of NGF HDL-mimicking nanoparticles prepared by an ion-pair strategy. NGF is a negatively charged hydrophilic molecule. A cationic peptide, protamine, was used to neutralize the charges and formed an ion-pair complex with NGF. Cholesteryl oleate, phospholipids, and TPGS formed self-assembly prototype NPs by homogenization. The NGF/protamine complex was incorporated into the prototype NPs. Finally, Apo A-I was coated on the NP surface after overnight incubation. This figure has been modified from Prathipati et al.16. Please click here to view a larger version of this figure.
Figure 2: The influence of homogenization on the particle size of the prototype nanoparticles. The excipients, PC, SM, PS, CO, and TPGS, which were dissolved in ethanol, were added to glass vials, and the solvent was evaporated under N2 stream. 1 mL of water was added and homogenized at 9,500 rpm for different times. The particle sizes of subsequent nanoparticles were measured. Data are presented as the mean ± standard deviation (n = 4). This figure has been modified from Prathipati et al.16. Please click here to view a larger version of this figure.
Figure 3: Particle size and size distribution of blank HDL-mimicking nanoparticles and NGF HDL-mimicking nanoparticles. The particle sizes and distributions were measured using a particle analyzer. This figure has been modified from Prathipati et al.16. Please click here to view a larger version of this figure.
Figure 4: Chromatograms of free NGF and NGF HDL-mimicking nanoparticles on a Sepharose CL-4B column eluted by PBS. 200 µL of free NGF solution (10 µg/mL) and NGF NP solution were loaded onto the gel filtration column and eluted with 1x PBS. A total of twelve fractions (1 mL for each fraction) were collected for both samples. The NP intensity in each fraction was measured by a particle analyzer, and the concentration of NGF in each fraction was measured using a Sandwich ELISA kit. This figure has been modified from Prathipati et al.16. Please click here to view a larger version of this figure.
Figure 5: In vitro release of NGF HDL-mimicking nanoparticles measured by a dialysis method. PBS with 5% BSA was used as a release medium. 200 µL of free NGF solution (10 µg/mL) or NGF NPs were added to a dialysis tube supplemented with 400 µL of the release medium. The dialysis tube was put into 30 mL of pre-warmed release medium. The study was performed at 37 °C with 135 rpm shaking. At 1, 2, 4, 6, 8, 24, 48, and 72 h, 100 µL of the release medium was withdrawn and replaced with 100 µL of fresh medium. The NGF concentration in each sample was measured using a Sandwich ELISA kit. Data are presented as the mean ± standard deviation (n = 4). This figure has been modified from Prathipati et al.16. Please click here to view a larger version of this figure.
Figure 6: The influence of NGF HDL-mimicking nanoparticles on neurite outgrowth in PC12 cells. Cells were treated with 50 ng/mL free NGF (A) and NGF HDL-mimicking nanoparticles (B) for 7 days. The neurite was imaged using an inverted light microscope under 10X magnification. Please click here to view a larger version of this figure.
Figure 7: The comparison of biodistribution between free NGF and NGF HDL-mimicking nanoparticles in mice (n = 3). The mice were administered with 40 mg/kg of NGF by tail-vein injection and were sacrificed at 30 min after administration. The blood, liver, spleen, and kidney were collected, and the concentration of NGF in each sample was measured using a Sandwich ELISA kit. Data are shown as the mean ± standard deviation. This figure has been modified from Prathipati et al.16. Please click here to view a larger version of this figure.
Sample | Particle size (nm) | P.I. | EE% of NGF | Zeta potential (mV) |
NGF HDL-mimicking NPs | 171.4 ±6.6 | 0.239 ±0.01 | 65.9 ±1.4 | -12.5 ±1.9 |
Table 1: Characterization of NGF HDL-mimicking nanoparticles (n = 3). Data are shown as the mean ± standard deviation. This table has been modified from Prathipati et al.16.
Recombinant Human Beta-NGF | Creative Biomart | NGF-05H | |
L-a-Phosphatidylcholine (PC) | Avanti | 131601P | 95%, Egg, Chicken |
Sphingomyelin (SM) | Avanti | 860062P | Brain, Porcine |
Phosphatidylserine (PS) | Avanti | 840032P | Brain, Porcine |
Cholesteryl oleate (CO) | Sigma | C9253 | |
D-α-Tocopheryl polyethylene glycol succinate (TPGS) | BASF | 9002-96-4 | Vitamin E Polyethylene Glycol Succinate |
Protamine sulfate | Sigma | P3369 | meets USP testing specifications |
Apolipoprotein A1, Human plasma | Athens Research & Technology | 16-16-120101 | 1mg in 671 µl 10 mM NH4HCO3, pH 7.4 |
Sepharose 4B-CL | Sigma | CL4B200 | Cross-linked agarose, gel filtration chromatography column filling material |
Sandwich ELISA Kit for NGF | R&D system | DY008 | |
Bovine Serum Albumin | Sigma | A2153 | |
RPMI-1640 medium | GE Healthcare Life Science | SH30096.02 | |
Horse serum | GE Healthcare Life Science | SH30074.03 | |
Fetal bovine serum | Gibco | 10082147 | |
PC12 cells | ATCC | CRL-1721 | |
Rat tail collagen type I | Sigma | C3867 | |
Sodium acetate | Sigma | S2889 | |
Sodium chloride | Sigma | 31414 | |
Triton X-100 | Sigma | T8787 | |
Phenylmethanesulfonyl fluoride (PMSF) | Sigma | P7626 | |
Benzethonium chloride | Sigma | B8879 | |
Name | Company | Catalog Number | Comments |
Equipment | |||
Homogenizer | Tekmar | T 25-S1 | |
Delsa Nano HC particle analyzer | Beckman-Coulter | Delsa Nano HC | |
Float-A-Lyzer G2 Dialysis Device | Spectrum Laboratories | G235036 | Molecule Cutoff 300 kDa |
Centrifuge | Eppendoff | 5424R | |
Polytron homogenizer | Kinematica | PT 1200C | |
DecapiCone | Braintree Scientific Inc. | DC-M200 |
The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein (HDL)-mimicking nanoparticles (NPs). HDLs are endogenous NPs and have been explored as vehicles for the delivery of therapeutic agents. Various methods have been developed to prepare HDL-mimicking NPs. However, they are generally complicated, time consuming, and difficult for industrial scale-up. In this study, one-step homogenization was used to mix the excipients and form the prototype NPs. NGF is a water-soluble protein of 26 kDa. To facilitate the encapsulation of NGF into the lipid environment of HDL-mimicking NPs, protamine USP was used to form an ion-pair complex with NGF to neutralize the charges on the NGF surface. The NGF/protamine complex was then introduced into the prototype NPs. Apolipoprotein A-I was finally coated on the surface of the NPs. NGF HDL-mimicking NPs showed preferable properties in terms of particle size, size distribution, entrapment efficiency, in vitro release, bioactivity, and biodistribution. With the careful design and exploration of homogenization in HDL-mimicking NPs, the procedure was greatly simplified, and the NPs were made scalable. Moreover, various challenges, such as separating unloaded NGF from the NPs, conducting reliable in vitro release studies, and measuring the bioactivity of the NPs, were overcome.
The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein (HDL)-mimicking nanoparticles (NPs). HDLs are endogenous NPs and have been explored as vehicles for the delivery of therapeutic agents. Various methods have been developed to prepare HDL-mimicking NPs. However, they are generally complicated, time consuming, and difficult for industrial scale-up. In this study, one-step homogenization was used to mix the excipients and form the prototype NPs. NGF is a water-soluble protein of 26 kDa. To facilitate the encapsulation of NGF into the lipid environment of HDL-mimicking NPs, protamine USP was used to form an ion-pair complex with NGF to neutralize the charges on the NGF surface. The NGF/protamine complex was then introduced into the prototype NPs. Apolipoprotein A-I was finally coated on the surface of the NPs. NGF HDL-mimicking NPs showed preferable properties in terms of particle size, size distribution, entrapment efficiency, in vitro release, bioactivity, and biodistribution. With the careful design and exploration of homogenization in HDL-mimicking NPs, the procedure was greatly simplified, and the NPs were made scalable. Moreover, various challenges, such as separating unloaded NGF from the NPs, conducting reliable in vitro release studies, and measuring the bioactivity of the NPs, were overcome.
The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein (HDL)-mimicking nanoparticles (NPs). HDLs are endogenous NPs and have been explored as vehicles for the delivery of therapeutic agents. Various methods have been developed to prepare HDL-mimicking NPs. However, they are generally complicated, time consuming, and difficult for industrial scale-up. In this study, one-step homogenization was used to mix the excipients and form the prototype NPs. NGF is a water-soluble protein of 26 kDa. To facilitate the encapsulation of NGF into the lipid environment of HDL-mimicking NPs, protamine USP was used to form an ion-pair complex with NGF to neutralize the charges on the NGF surface. The NGF/protamine complex was then introduced into the prototype NPs. Apolipoprotein A-I was finally coated on the surface of the NPs. NGF HDL-mimicking NPs showed preferable properties in terms of particle size, size distribution, entrapment efficiency, in vitro release, bioactivity, and biodistribution. With the careful design and exploration of homogenization in HDL-mimicking NPs, the procedure was greatly simplified, and the NPs were made scalable. Moreover, various challenges, such as separating unloaded NGF from the NPs, conducting reliable in vitro release studies, and measuring the bioactivity of the NPs, were overcome.