Here, we present a protocol for the synthesis of placental chondroitin sulfate A binding peptide (plCSA-BP)-conjugated lipid-polymer nanoparticles via single-step sonication and bioconjugate techniques. These particles constitute a novel tool for the targeted delivery of therapeutics to most human tumors and placental trophoblasts to treat cancers and placental disorders.
An effective cancer therapeutic method reduces and eliminates tumors with minimal systemic toxicity. Actively targeting nanoparticles offer a promising approach to cancer therapy. The glycosaminoglycan placental chondroitin sulfate A (plCSA) is expressed on a wide range of cancer cells and placental trophoblasts, and malarial protein VAR2CSA can specifically bind to plCSA. A reported placental chondroitin sulfate A binding peptide (plCSA-BP), derived from malarial protein VAR2CSA, can also specifically bind to plCSA on cancer cells and placental trophoblasts. Hence, plCSA-BP-conjugated nanoparticles could be used as a tool for targeted drug delivery to human cancers and placental trophoblasts. In this protocol, we describe a method to synthesize plCSA-BP-conjugated lipid-polymer nanoparticles loaded with doxorubicin (plCSA-DNPs); the method consists of a single sonication step and bioconjugate techniques. In addition, several methods for characterizing plCSA-DNPs, including determining their physicochemical properties and cellular uptake by placental choriocarcinoma (JEG3) cells, are described.
An effective cancer therapeutic method reduces and eliminates tumors with minimal systemic toxicity. Hence, selective tumor targeting is the key to exploring successful therapeutic methods. Nanoparticles offer a promising opportunity for cancer therapy, and molecular assemblies with different functional groups will enhance drug efficacy and reduce associated side effects1,2. Moreover, nanoparticle systems mainly utilize passive and active targeting to reach target tumors3.
Passive targeting exploits the innate characteristics of nanoparticles and enhanced permeability and retention (EPR) effects to reach tumor cells. Cationic liposomes have been successfully used to deliver various anticancer drugs to tumors in clinical applications4,5,6. Despite the potential effective cancer therapeutic effect, a low drug concentration in the tumor region and an inability to distinguish tumor cells from normal tissues are two major limitations of passive-targeting nanoparticles7.
Active targeting strategies take advantage of antigen-antibody, ligand-receptor and other molecular recognition interactions to specifically deliver drugs to tumors8. The glycosaminoglycan placental chondroitin sulfate A (plCSA) is broadly expressed on most cancer cells and placental trophoblasts. Moreover, the malarial protein VAR2CSA can specifically bind to plCSA9,10. Hence, VAR2CSA can be a tool for targeting human cancer cells. However, when VAR2CSA is conjugated to nanoparticles, the full-length protein may limit the penetration of nanoparticles into tumor cells. Recently, we discovered a plCSA binding peptide (plCSA-BP), derived from the malarial protein VAR2CSA. plCSA-BP-conjugated lipid-polymer nanoparticles rapidly bonded to choriocarcinoma cells and significantly increased doxorubicin (DOX) anticancer activity in vivo11; these particles also specifically bonded to placental trophoblasts and could serve as a tool for the targeted delivery of drugs to the placenta12.
Lipid-polymer nanoparticles consist of a lipid monolayer shell and a hydrophobic polymeric core and represent a new carrier for drug delivery. These nanoparticles combine the advantages of liposomes and polymeric nanocarriers, such as controllable nanoparticle size, high biocompatibility, sustained drug release, high drug loading efficiency (LE), and excellent stability13. In this work, we used a single-step sonication method to synthesize lipid-polymer nanoparticles. This method is fast, convenient and suitable for scale-up and has been widely used to prepare lipid-polymer nanoparticles by our group11,14 and others15,16,17,18.
1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) is a popular carbodiimide used as a crosslinking agent for conjugating biomolecules containing amines and carboxylates19. In addition to EDC, N-hydroxysulfosuccinimide (NHS) is the most common conjugation reagent in surface and nanoparticle conjugation reactions20,21. NHS can reduce the number of side reactions and enhance the stability and yield of ester intermediates22,23.
Here, we describe a protocol for synthesizing plCSA-targeted lipid-polymer nanoparticles. First, the single-step sonication synthesis of DOX-loaded lipid-polymer nanoparticles (DNPs) is described. Then, an EDC/NHS bioconjugate technique for generating plCSA-BP-conjugated lipid-polymer nanoparticles is introduced. This bioconjugate technique can also be used to conjugate other antibodies and peptides to nanoparticles. Finally, we describe the physicochemical properties and in vitro assay used to characterize the plCSA-targeted lipid-polymer nanoparticles. We believe that these plCSA-targeted lipid-polymer nanoparticles could constitute an effective system for the targeted delivery of drugs to most human cancers and the targeted delivery of payloads to the placenta to treat placental disorders.
1. Preparation of Stock Solutions
2. Synthesis of DNPs
NOTE: To avoid DOX photochemical degradation, all operations were performed in the dark.
Nanoparticles were synthesized by a previously reported single-step sonication method11,13,14.
3. Conjugation of Peptides to DNPs
4. Characterization of plCSA-Targeted Lipid-Polymer Nanoparticles
Vial | Volume of diluent (μL) | Volume and source of peptide (μL) | Final peptide concentration (μg/mL) |
A | 0 | 300 of Stock | 1000 |
B | 250 | 250 of vial A dilution | 500 |
C | 250 | 250 of vial B dilution | 250 |
D | 250 | 250 of vial C dilution | 125 |
E | 300 | 200 of vial D dilution | 50 |
F | 250 | 250 of vial E dilution | 25 |
G | 400 | 100 of vial F dilution | 5 |
H | 500 | 0 | 0 |
Table 1. Preparation of Standard peptides
5. Fluorescence Microscopy Assessment of plCSA-Targeted Nanoparticle Uptake in Choriocarcinoma (JEG3) Cells
In this protocol, PLGA, DSPE-PEG-COOH and soybean lecithin are a representative polymer, lipid-PEG-COOH conjugate and lipid, respectively. The synthesis of plCSA-targeted lipid-polymer nanoparticles via a single-step sonication method and an EDC/NHS technique is illustrated in Figure 1. First, under sonication conditions, soybean lecithin, PLGA and DSPE-PEG-COOH self-assemble to form core-shell structured DNPs. The core consists of PLGA and encapsulated DOX, the shell consists of DSPE-PEG-COOH, and the soybean lecithin monolayer is located at the interface of the shell and the core. Then, the -COOH groups exposed on the DNP surface were conjugated with the -NH2 groups of the peptide via the EDC/NHS technique to synthesize plCSA-DNPs.
The DNPs prepared in this protocol were 82.3 ± 4.7 nm in diameter, and after conjugation to plCSA-BP, the diameter of the primary DNPs increased to 109.3 ± 5.9 nm (Figure 2). The PDI of the DNPs and plCSA-DNPs was 0.127±0.005 and 0.134 ± 0.065, respectively. TEM images of the DNPs and plCSA-DNPs also showed that particles were well dispersed and generally had spherical morphologies (Figure 3). The zeta potential of the DNPs and plCSA-DNPs was -20.1 ± 1.32 mV and -29.9 ± 3.56 mV, respectively (Table 2). Hence, these nanoparticles are predicted to be highly stable.
Nanoparticles | Diameter(nm) | PDI | Zeta potential (mV) |
DNPs | 82.3±4.7 | 0.127±0.005 | -20.1±1.32 |
plCSA-DNPs | 109.3±5.9 | 0.134±0.065 | -29.9±3.56 |
PDI:polydispersity index. Data are presented mean ± SD (n = 3). |
Table 2. Characterization of nanoparticles
The EE and LE of nanoparticles are important for clinical applications. The EE of DOX by the DNPs and plCSA-DNPs was 40.3 ± 1.67% and 39.5 ± 1.94%, respectively. The LE of DOX in the DNPs and plCSA-DNPs was 6.2 ± 0.74% and 5.1 ± 0.42%, respectively. These data showed that the extent of drug loading and drug encapsulation was maintained after plCSA-BP decoration. The decoration efficiency of plCSA-BP on the surface of DNPs was determined by the BCA assay (Figure 4). The plCSA-BP conjugation efficiency was found to be 45.5 ± 3.7%.
The JEG3 cellular uptake assay indicated that the plCSA-DNPs rapidly bound to JEG3 cells within 30 min (Figure 5). Therefore, plCSA-BP was efficiently conjugated to the DNP surface in this protocol. Moreover, plCSA-BP could rapidly bind to JEG3 cells and increase the cellular uptake of the nanoparticles.
Figure 1. Schematic illustration of the method used to synthesize plCSA-targeted lipid-polymer nanoparticles. First, a single-step sonication method was used to synthesize lipid-polymer nanoparticles (DNPs), and then the EDC/NHS technique was used to conjugate peptides to the nanoparticle surfaces (plCSA-DNPs). Please click here to view a larger version of this figure.
Figure 2. Size distribution of the different nanoparticles. Hydrodynamic size of DNPs (A) and plCSA-DNPs (B) measured by DLS. Representative figures are presented to demonstrate the particle size and size distribution. Please click here to view a larger version of this figure.
Figure 3. Nanoparticle morphology characterized by TEM. Typical TEM image of DNPs (A) and plCSA-DNPs (B) observed with 2% phosphotungstic acid staining. Scale bar=100 nm. Please click here to view a larger version of this figure.
Figure 4. Measurement of the conjugation efficiency using the BCA assay. Standard curve of the peptides at 562 nm. Please click here to view a larger version of this figure.
Figure 5. Nanoparticle uptake by JEG3 cells. JEG3 cells were analyzed by fluorescence microscopy after 30 min of incubation with 5 µg/mL different nanoparticles. Red: DOX, blue: DAPI-labeled nuclei. Scale bar=50 µm. Please click here to view a larger version of this figure.
This protocol provides an efficient and reproducible method for synthesizing plCSA-BP-conjugated lipid-polymer nanoparticles. The single-step sonication method to prepare lipid-polymer nanoparticles is fast, reproducible and different from typical nanoprecipitation methods that involve heating, vortexing, or evaporation. Hence, the developed method significantly reduces the synthesis time. In addition, the EDC/NHS bioconjugate used in this protocol is a commonly used and convenient technique to conjugate peptides and antibodies to nanoparticles. Therefore, the combination of the single-step sonication method with the EDC/NHS bioconjugate technique to synthesize plCSA-targeting lipid-polymer nanoparticles can be scaled up for clinical applications.
Certain procedures are critical for successfully synthesizing and characterizing plCSA-DNPs. First, the relative concentrations of lecithin, PLGA and DSPE-PEG-COOH determine the lipid-polymer nanoparticle size and polydispersity. When the concentration of lecithin is fixed, a higher concentration of DSPE-PEG-COOH results in lower polydispersity and a smaller nanoparticle size,13,15 which consequently decreases the drug LE. When the concentration of PLGA is fixed, more DSPE-PEG-COOH is replaced by lecithin, leading to higher nanoparticle polydispersity and larger nanoparticle size15. In this situation, the nanoparticles are more unstable and aggregate easily. In this protocol, the DSPE-PEG-COOH/PLGA weight ratio is 0.11, and the lecithin/DSPE-PEG-COOH mass ratio is 0.43. The lipid-polymer nanoparticle size is approximately 82 nm with a PDI of approximately 0.127.
Second, the pH of the solution during the conjugation of the peptides to the nanoparticles is of utmost importance. The optimal pH of ester activation is 5.0-6.0, and the amine reaction is most efficient at pH 7.2-7.519,28,29. For best results, the reaction should be performed in two steps. First, ester activation occurs in MES (or another noncarboxylate, nonamine buffer) at pH 5.0-6.0. Then, the pH is adjusted to 7.2-7.5 using PBS (or another nonamine buffer) immediately before the reaction with the amine-containing molecule19. Considering that the half-life of NHS esters is 4-5 h at pH 7.019, the duration of the amine reaction exceeds 10 h at 4 °C.
The final critical factor is nanoparticle uptake. Nontargeted uptake of DNPs by JGE3 cells was eliminated in a function analysis of the targeted uptake by controlling the culture time and temperature and the concentration of FBS in the medium. In this protocol, JEG3 cells were incubated with 5% FBS at 4 °C for 1 h, and then cultured at 37 °C for 30 min to minimize DNP cellular uptake.
If issues arise during the procedure, there are three major troubleshooting steps related to the synthesis and characterization of plCSA-DNPs. First, lipid-polymer nanoparticles were prepared by the self-assembly of PLGA and lecithin under sonication conditions. PLGA only dissolves in organic solvent. Hence, upon addition to a hydrophilic solvent, PLGA precipitates quickly into small nanoparticles. To minimize the effect of the speed of the dropwise addition and avoid generating larger particles, sonication of the mixture for 1-2 min before adding PLGA is necessary. Second, although quantitative analysis of the plCSA-BP conjugation efficiency using the BCA method is quick and convenient, a high-performance liquid chromatography (HPLC) assay is more accurate. The HPLC experimental details have been described by our group11 and others30,31,32. Finally, the optimal conditions for plCSA-DNP cellular uptake may differ for other cancer cells. Gradually decreasing the FBS concentration and increasing incubation time at 37 °C may help determine the optimal conditions.
One limitation of the protocol is that the hydrophobicity of drugs determines the EE of the drugs. Compared with hydrophilic drugs, hydrophobic drugs have a stronger affinity for PLGA, which results in an increased drug EE. In these instances, the procedures for the synthesis of the plCSA-targeted lipid-polymer nanoparticles will have to be examined experimentally to determine the most efficient synthesis method that results in the ideal drug LE and EE.
As mentioned above, the glycosaminoglycan plCSA is specifically expressed on most cancer cells and placental trophoblasts. Hence, it is important to note that the plCSA-NPs should be used for the targeted delivery of therapeutics to tumor cells in only nonpregnant animals and humans, and that these drugs-loaded particles would lead to side effects in the placenta of pregnant animals and humans.
In summary, we have described a simple and convenient method to synthesize plCSA-targeted lipid-polymer nanoparticles. The significance of this protocol is that it increases the drug availability at tumors and the placenta, while minimizing the concomitant toxicity. This approach should be useful for the targeted delivery of drugs to tumors and placenta and may be scaled up to prepare plCSA-targeted lipid-polymer nanoparticles for clinical applications in the future.
The authors have nothing to disclose.
This work was supported by grants from the National Key Research and Development Program of China (2016YFC1000402), the National Natural Sciences Foundation (81571445 and 81771617) and the Natural Science Foundation of Guangdong Province (2016A030313178) to X.F. and the Shenzhen Basic Research Fund (JCYJ20170413165233512) to X.F.
plCSA peptide | Shanghai GL Biochem | 573518 | for peptide synthesis |
Ethanol absolute | Sinopharm Chemical | 10009218 | for nanoparticles synthesis |
Soybean lecithin | Avanti Polar Lipids | 441601 | for nanoparticles synthesis |
DSPE-PEG-COOH | Avanti Polar Lipids | 880125 | for nanoparticles synthesis |
Doxorubicin | JKChemical | 113424 | for nanoparticles synthesis |
Acetonitrile | Shanghai Lingfeng | 1008621 | for nanoparticles synthesis |
PLGA | Sigma-Aldrich | 719897 | for nanoparticles synthesis |
Ultrasonic processor | Sonics | VCX130 | for nanoparticles synthesis |
Centrifuge filter (MWCO 10 kDa) | Millipore | UFC801024 | for nanoparticles purification |
centrifuge | Sigma | 3-18KS | for nanoparticles purification |
2-[morpholino]ethanesulfonic acid(MES) | Sigma-Aldrich | M3671 | for peptide conjugation |
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) | Sigma-Aldrich | 3450 | for peptide conjugation |
N-hydroxysuccinimide (NHS) | Sigma-Aldrich | 56480 | for peptide conjugation |
Dialysis bags | Spectrum | 132592T | for nanoparticles purification |
PBS | Hyclone | SH30028.01 | for cell culture |
10 mL centrifuge tubes, polypropylene | Aladdin | S-025 | for nanoparticles synthesis |
15 mL centrifuge tubes, polypropylene | Corning | 430791 | for various applications |
0.22 μm sterile syringe filter | Millipore | SLGV033RB | for nanoparticles purification |
1 ml syringe, polypropylene | BD | 328421 | for nanoparticles synthesis |
Malvern Zetasizer | Malvern | Nano ZS | for particle size analyer |
Phosphotungstic acid | for TEM | ||
TEM grid | EMCN | BZ10024a | for TEM |
UV-VIS spectrometer | Leagene | DZ0035 | for TEM |
Transmission electron microscope |
JEOL | JEM-100CXII | for particle size analyer |
BCA reagent A | Thermo Fisher Scientific | 23228 | for BCA assay |
BCA reagent B | Thermo Fisher Scientific | 23224 | for BCA assay |
96-Well Plates | Corning | 3599 | for BCA assay |
Plate reader | Thermo Fisher Scientific | Multiskan™ GO | for BCA assay |
12-well plates | Corning | 3513 | for cell culture |
JEG3 cell | Cell Bank of the Chinese Academy of Sciences | TCHu195 | Human placenta |
DMEM/F12 | Hyclone | SH30272.01 | phenol red-free |
Fetal bovine serum (FBS) | GIBCO | 10100 | for cell culture |
Penicillin/streptomycin | GIBCO | 15070063 | for cell culture |
Fluorescence microscope | OLYMPUS | CKK53 | for celluar uptake |
Paraformaldehyde | Shanghai Lingfeng | 1372021 | for celluar uptake |
DAPI | Sangon Biotech | A606584 | for celluar uptake |
Mounting medium | Life | P36961 | for celluar uptake |