Size control of lipid-based drug carrier by drug loading

Abstract

A phospholipid–protein hybrid nanostructure was found to reveal various hydrodynamic diameters dependent on the amount of the drugs incorporated, which could be over 10 times larger than that of the starting nanostructure. This observation was likely related to the thermal stabilization of the nanostructure during incorporation of the drugs by using the increased amount of phospholipids for the preparation. Furthermore, hydrophobicity or molecular weight of drugs is at least needed for size control.

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Cancer nanotechnology is a multidisciplinary research area that covers the design and engineering of nanostructures for cancer imaging and therapy, and high-specificity detection of cancer-related DNA and protein.¹ Mesoscale nanoparticles (5–100 nm in diameter) designed to be biocompatible are attractive tools for drug delivery. This is because such nanoparticles can passively target tumor tissues and inflammatory sites without any molecular recognition elements like antibodies.² Blood vessels in tumor tissues and inflammatory sites are leaky, which enables the nanoparticles to extravasate at their sites. Among such nanoparticles, a liposome loaded with doxorubicin (DXR), an anticancer drug, has been already clinically used as Doxil.³ Doxil consists of polyethylene glycol (PEG)-grafted unilamellar vesicles with an approximate mean size of 100 nm. PEG conjugation protects the liposome from rapid recognition and clearance by macrophages in the liver and spleen. Whereas the size and stealth nature of Doxil are suitable for passive targeting to tumor tissues, PEG-liposomes potentially elicit a strong immune response through abundant production of PEG-specific IgM, which causes rapid clearance of their second dose from the circulation.⁴

Uni/multilamellar vesicles or micelles prepared with phospholipids react with a serum protein, apolipoprotein A-I (apoA-I) to form a discoidal lipid bilayer circumscribed by apoA-I (Fig. 1), named nascent high density lipoprotein (HDL). Nascent HDLs occupy 5–10% of total HDL in normal human plasma. Thus, much attention has been focused recently on the use of reconstituted nascent HDLs as a drug carrier.^5,6 Nevertheless, their intrinsic size (∼10 nm) may not be advantageous for application in drug delivery. Size control of nascent HDLs in the range of 10–50 nm was recently reported,^7,8 yet, to our knowledge, no prior study has shown the preparation of mesoscale nascent HDLs by drug loading. In this study, we describe the first facile method for the size control of nascent HDL as a biocompatible lipid-based drug carrier.

Fig. 1 Schematic illustration of drug-loaded nascent HDL preparation. Based on the ΔapoA-I crystal structure, reconstituted nascent HDL is proposed to adopt structures in which a pseudo-continuous amphipathic α-helix of apoA-I wraps around the edge of the phospholipid bilayer disc.

Nascent HDL was reconstituted with palmitoyl-oleoyl phosphatidylcholine (POPC) and an NH₂-terminal 43 amino acid deletion mutant of 243 amino acid human apoA-I (ΔapoA-I), which was expressed in E. coli, in a 250 : 1 molar ratio. This molar ratio is relatively high compared with 100 : 1 for the preparation of nascent HDL generally used for the biochemical studies.⁹ The deletion of apoA-I is due to general advantage of lower molecular weight proteins for efficient E. coli expression. This deletion mutant is reported to adopt an open-ring circular shape (Fig. 1),¹⁰ which is assumed to be indicative of the lipid-bound structure of apoA-I in nascent HDLs, while the parent apoA-I possesses an α-helical compact structure.¹¹ The mean hydrodynamic diameter of nascent HDL reconstituted with the deletion mutant (rΔHDL) was 19 nm. Upon reconstitution in a 100 : 1 molar ratio, the diameter of rΔHDL was 13 nm, which was well correlated with the Stokes’ diameters (8.5–16.5 nm) determined by native gradient gel electrophoresis.⁹

This slightly enlarged rΔHDL was first characterized using DXR to assess the efficiency of drug loading. The loading reaction was carried out by incubation of rΔHDL with DXR for 0.5, 1, or 1.5 h at 37, 50, or 60 °C, and the reaction mixture was passed through gel filtration chromatography to remove free DXR and insoluble aggregates generated during the reaction. Fig. 2A displays the hydrodynamic diameter of these DXR-loaded rΔHDLs (DXR-rΔHDLs) in dynamic light scattering (DLS) analysis. It is noteworthy that DXR-rΔHDLs exhibited a diversity of mean diameter, which ranged from 16 to 232 nm (Fig. 2A). It appears that the value is dependent on the amount of DXR in each DXR-rΔHDL (Fig. 2B). To confirm this, the volume of each DXR-rΔHDL was estimated on the basis of the mean diameter and plotted against the amount of DXR (ESI, Fig. S1†). A linear relationship between them was found (r = 0.988), which indicates that rΔHDL is inflated by incorporating DXR. This hypothesis was supported by the observation that heat treatment of rΔHDL alone (50 °C for 1 h and 60 °C for 1.5 h) causes at most a 20% increase in diameter. This dramatic enlargement of nascent HDL by DXR loading would be structurally possible, because the number of apoA-I in nascent HDL is variable and increases with increasing the diameter.⁸ To our knowledge, this is the first observation of the size control of lipid-based drug carriers by the carried drug over a dynamic range of 2 orders of magnitude.

Fig. 2 Size control of rΔHDL by DXR loading under various conditions. Reaction temperature- and time-dependent increases in the diameter of the complex (A) and the amount of DXR incorporated into rΔHDL (B) were observed for the gel filtration column eluates from the reaction mixture containing rΔHDL (0.1 mg mL⁻¹ on a protein basis) and DXR (0.4 mg mL⁻¹). rΔHDL was incubated with DXR in an aqueous solution for 0.5, 1, or 1.5 h at 37, 50, or 60 °C. The numbers in each panel in A denote the mean diameter determined by DLS analysis. Averages of data for two experiments were shown.

The recovery rate of rΔHDL during the loading reaction was determined to be approximately 90% for each condition based on densitometric analysis of gel bands from each DXR-rΔHDL eluate (ESI, Fig. S2†). This result suggests that rΔHDL can be stably loaded with DXR even at 60 °C without significant thermal denaturation-induced aggregate formation. It was also confirmed that the high DXR loading capacity and thermal stability of rΔHDL resulted from the use of 250 mol equiv. of POPC (ESI, Fig. S3†).

Next, we examined the applicability of this size control strategy to other drugs. cis-Diamineplatinum(II) dichloride (CDDP), trichostatin A (TSA), zinc phthalocyanine (ZnPc), dexamethasone (DEX), 5-fluorouracil (5-FU), or all-trans retinoic acid (ATRA) was incubated with rΔHDL. For these drug loadings, two incubation conditions (50 °C for 1 h and 60 °C for 1.5 h) were selected because drug loading in the former condition was successful for DXR and large increases in the DXR loading amount (17 → 61 μg) and the diameter (155 → 232 nm) were observed in the latter condition compared with in the former one (Fig. 2). We expected here that the comparison of the results between the two conditions enables assessment of drug loading as well as loading amount-dependent enlargement. As shown in Fig. 3A, slight and marked increases in diameter after the reaction at 50 °C for 1 h were observed for TSA and ZnPC, respectively. The diameter was further increased in the reaction with TSA at 60 °C for 1.5 h.

Fig. 3 Size control of rΔHDL by altering drug loading. Loading reactions were performed at 50 °C for 1 h or at 60 °C for 1.5 h. Except for ZnPc (0.04 mg mL⁻¹) and ATRA (0.04 mg mL⁻¹), the drug concentration in the reaction mixture was 0.4 mg mL⁻¹. DLS histograms (A) and UV/vis absorption spectra (B) of each gel filtration column eluate were shown. The numbers in each panel in (A) denote the mean diameter. In (B), only the absorption range of each drug incorporated is shown, and the black spectrum in each panel corresponds to rΔHDL. Representative data for two experiments are shown.

Fig. 3B depicts the absorption spectra of the drug-loaded rΔHDLs. The absorptions of TSA around 350 nm and ZnPc between 300–700 nm were evident in each spectrum, implying their stable incorporation into rΔHDL. The amount of TSA in rΔHDL increased by prolongation of the reaction time and elevation in the reaction temperature. Therefore, TSA was also found to enlarge rΔHDL in a loading amount-dependent manner like DXR. On the other hand, ZnPc revealed no significant increase in the loading amount. During the gel filtration process (the exclusion limit of 5 kDa), blue ZnPc in both the reaction solutions was completely eluted with rΔHDL. In the absence of rΔHDL, no ZnPc was eluted owing to entrapment by the column (data not shown). These results indicate that, since all ZnPc added was incorporated into rΔHDL in both the reaction conditions, there was no difference in the loading amount. In terms of the loading amount-dependent enlargement, this consideration may rationalize no change in the diameter between the two conditions. A complete incorporation of the drug added would occur for DEX and ATRA, considering that the spectra exhibiting their incorporation were almost the same between the two conditions.

To further verify the correlation, we examined the relationship between the diameter and the loading amount of the drugs by altering the drug concentration in the reaction solutions. As illustrated in Fig. 4B, each loading amount of the four drugs was dynamically changed in a drug concentration-dependent manner. From the absorbance, they were found to be approximately proportional to the initial concentration of the drug. The diameter of rΔHDL loaded with ZnPc or ATRA increased clearly with increasing the amount of the drugs incorporated. In contrast, the diameters of TSA- and DEX-loaded rΔHDL were enlarged slightly, and they seemed to reach a plateau at ∼40 nm. The profile of the drug loading-dependent diameter change for the 7 drugs used in this study is likely related to their partition coefficients (log p) (ESI, Fig. S4†). At least, no obvious incorporation of CDDP and 5-FU is explained by their low values (log p < 0). This implies that they are too hydrophilic to be incorporated into the hydrophobic lipid core of rΔHDL. On the other hand, both ZnPc and ATRA, which were incorporated well and showed the loading amount-dependent enlargement, have the high values (log p > 6). In the case of TSA and DEX with the middle value (log p = 1–3), their incorporation was successful but the diameters were not dependent on the loading amount. However, this assumption is not fully applicable to the results for DXR with the middle value showing the size control over a wide range. Thus, other factors beside hydrophobicity would also be considered. When comparing the molecular weight of the 7 drugs, CDDP and 5-FU, showing no obvious incorporation, have a relatively low molecular weight (≤300), while ZnPc and DXR, inducing the loading amount-dependent enlargement, have a high molecular weight over 500 (ESI, Fig. S4†). The molecular weights of TSA and DEX are between the two groups. This time, however, the results for ATRA (molecular weight 300), showing the size control, did not conform this consideration. In light of these discussions, the size control of rΔHDL by drug loading likely arises with the use of hydrophobic or high-molecular weight drugs.

Fig. 4 Size control of rΔHDL by altering the drug concentration. The loading reaction was performed at 60 °C for 1.5 h at a concentration of 0.4, 0.8, and 1.6 mg mL⁻¹ (TSA), 0.02, 0.04, and 0.08 mg mL⁻¹ (ZnPc), 0.4, 0.8, and 1.6 mg mL⁻¹ (DEX), and 0.04, 0.08, 0.16 mg mL⁻¹ (ATRA). DLS histograms (A) and UV/vis absorption spectra (B) of each gel filtration column eluate were shown. The numbers in black and the colored numbers in each panel in (A) denote the mean diameter and the drug concentration, respectively. The colored spectra in (B) were obtained for the eluates with the different drug concentration denoted by the same color in each panel. Representative data for two experiments were shown.

Conclusions

We have successfully developed a simple and unique method for controlling the size of nascent HDL within the mesoscale by incorporation of drugs for the first time. Although further studies are necessary to assess passive targeting of the incorporated drug after intravenous administration of the size-controlled rΔHDL, our finding raises the possibility of transforming nascent HDLs into a tumor and inflammatory site-targeted drug carrier by size control with the carried molecule.

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Footnote

† Electronic supplementary information (ESI) available: Methods, materials and equipment, and figures. See DOI: 10.1039/c001442g

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