Anal Bioanal Chem (2015) 407:6429–6434 DOI 10.1007/s00216-015-8805-0

RESEARCH PAPER

Rapid evaluation of the quantity of drugs encapsulated within nanoparticles by high-performance liquid chromatography in a monolithic silica column Naoki Itoh 1 & Tomofumi Santa 1 & Masaru Kato 1

Received: 13 March 2015 / Revised: 6 May 2015 / Accepted: 26 May 2015 / Published online: 14 June 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract Drug-containing nanoparticles, the foundation of nanomedicine, provide promise for the safe and effective delivery of drugs to their targets. In this study, we developed a simple method to determine the relative quantities of nanoparticle-encapsulated drugs by HPLC using a commercially available monolithic silica column. Amphotericin Band irinotecan-containing nanoparticles produced nearly simultaneous elution peaks (~7 min), suggesting that elution was largely driven by hydrodynamic effects and was relatively unaffected by differences in the encapsulated drug. A good correlation was observed between the intensity of the nanoparticle peak and the relative quantity of encapsulated drug. We used our method to characterize the effects of drug quantity and nanoparticle size on drug encapsulation rates within the nanoparticles. Encapsulation increased with increasing quantities of the drug in the preparation solution. This effect was greater for irinotecan than for amphotericin B. Although absolute encapsulation also increased with increasing nanoparticle size, encapsulation efficiency decreased. Thus, the monolith column is suitable for evaluating nanomedicine quality and may be used to evaluate many kinds of nanomaterials.

Keywords HPLC . Monolith . Nanoparticle . Drug . Nanomedicine

* Masaru Kato [email protected] 1

Graduate School of Pharmaceutical Sciences and GPLLI Program, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

Introduction Drug-containing nanoparticles (nanomedicines) are gaining attention as noninvasive therapeutic devices [1–4] because they can deliver an encapsulated drug and release it directly into the target organ or tissue. Nanomedicines are associated with fewer side effects, as they reduce the distribution of the drug to nontarget organs or tissues. The popularity and increasing use of nanoparticles has made it necessary to develop a simple and fast analytical method to ensure quality and safety [5–7]. These methods must address the quantity of nanoparticles in a preparation and determine how much drug has been encapsulated within those nanoparticles. Variations in drug encapsulation will alter the therapeutic activity of the nanomedicine, even if the number of nanoparticles is the same. For instance, the efficiency of drug delivery to the target tissue increases by a factor of 10 if the amount of drug within the nanomedicine also increases by 10. Therefore, it is important to verify how much drug is encapsulated within nanomedicines to ensure their safe and proper usage. Many methods for evaluating the encapsulated drugs have been reported [8–16], and several reviews have been published [17, 18]. Many simple methods do not require physical separation of the encapsulated and free drug [14–16]. These methods are based on spectroscopic techniques such as fluorescence, NMR, and ESR to detect signal differences between molecules encapsulated within nanoparticles and those that remain free in solution. Although these techniques are simple, they are also characterized by low sensitivity, limited applications, and the challenge presented by real biological samples that contain numerous interferents.

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To overcome these limitations, the nanoparticles are first separated from the other molecules in a solution. The encapsulated drug is then extracted by collapsing the nanoparticles and measured [8–13]. Although many nanoparticle purification methods (such as ultracentrifugation, dialysis, and ultrafiltration) have been developed, it remains difficult to remove interferents from the dispersed nanoparticle solution. The required pretreatments cause physicochemical changes that may result in the collapse of the nanoparticles or drug release during purification. Furthermore, these procedures are laborintensive and time-consuming. To overcome these defects, Yamamoto et al. developed a column switching HPLC system to analyze the amount of drug within nanoparticles without manual pretreatment [19]. The system separated nanoparticles and free drug first by a restricted access solid-phase extraction column; release of the encapsulated drug from the nanomedicine was performed by HPLC. Finally, the amount of released drug was determined on an analytical column. Although this method is reliable and effective, there remains a need for a simpler, faster analytical method. We have developed a simultaneous separation method for nanoparticles and small molecules on a commercially available monolithic silica column [20]. Simultaneous separation is accomplished by exploiting the bimodal structure, micrometersized flow-through pores, and nanometer-sized mesopores of the monolith. Nanoparticles are separated when they penetrate through the micrometer-sized flow-through pores because of the hydrodynamic effect [21], and small molecules are separated by the chromatographic interaction with the surface of the stationary phase in the mesopores. We have demonstrated the utility of this separation method and used it to evaluate nanoparticle purification methods and stability by quantification. If the composition of the nanoparticles does interfere with the detection of the encapsulated drug, it becomes possible to quantify the encapsulated drug by detection at the appropriate wavelength. We believe this method is suitable for evaluating the relative quantities of drug encapsulated within nanomedicines, an important factor in quality assurance. We prepared drug-containing silica nanoparticles and measured the amount of encapsulated drug by using a monolith column. We used the sol-gel reaction to prepare the drug-containing nanoparticles [22, 23]. Alkoxysilane was hydrolyzed and polymerized under moderate conditions to form siloxane bonds that produce a silica mesh structure that trapped molecules from a solution and formed a molecular encapsulated silica gel. Because the mesh structure is constructed and traps the molecule during the reaction, various kinds of molecules may be encapsulated within the silica network [24, 25]. Basic amino acids, such as arginine or lysine, have been added to the reaction solution to prepare silica nanoparticles [26, 27]. The size distribution of the nanoparticles prepared by this method was very narrow and could be controlled by altering the amount of alkoxysilane and reaction time. In this study, we examined the effect of drug type and amount, as well as the effect of

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nanoparticle size on the amount of encapsulated drug by using a monolith column.

Experimental methods Chemicals Amphotericin B was purchased from Wako Pure Chemical Industries (Osaka, Japan). Irinotecan hydrochloride trihydrate was obtained from Tokyo Chemical Industry (Tokyo, Japan). Other chemicals used were the same chemicals used in our previous paper [20]. Preparation of nanoparticles containing drug Silica nanoparticles containing drug were prepared by means of a reported method [20, 28]. Arginine (10 mg), amphotericin B (0.05–0.5 mg), or irinotecan (0.05–0.5 mg) was dissolved in 10 mL deionized water, and then 0.45 mL cyclohexane and 0.55 mL tetraethyl orthosilicate (TEOS) were added to the solution. The mixture was stirred at 60 °C for 20 h using a Teflon-coated magnetic stirring bar at 300 rpm, and nanoparticles with uniform size (about 20 nm) were prepared. The size of the nanoparticles increased when the amount of TEOS in the reaction solution was increased. HPLC analysis HPLC (Hitachi, Tokyo, Japan) analysis was performed by the same system we used in our previous paper [20]. The system consisted of two L-2160U LaChrom Ultra pumps, an L-2200U LaChrom autosampler, an L-2455U LaChrom diode array detector, an L-2485U LaChrom fluorescence detector, and an HPLC system organizer. Octadecyl-modified silica monolithic column (250 mm×3 mm, GL Sciences, Tokyo, Japan) was used. The surface area, flow-through size, mesopore size, and carbon content of the column were 200 m2/g, 2 μm, 18 nm, and 14 %, respectively. Mobile phase A was water containing a mixture of 200 mM formic acid and ammonium formate (pH 3.6), and mobile phase B was acetonitrile. The gradient elution program of the mobile phases was as follows: 68 % (A) from 0 to 25 min and then 68–54 % (A) from 25 to 35 min for amphotericin B- and rhodamine 110containing nanoparticles and 82 % (A) from 0 to 25 min and then 82–61 % (A) from 25 to 35 min for irinotecan-containing nanoparticle. The flow rates were 0.2 mL/min for 0–25 min and 0.5 mL/min after 25 min. The injection volume was 10 μL, and a diode array detector and a fluorescence detector (excitation 365 nm, emission 440 nm for irinotecan and excitation 480 nm, emission 520 nm for rhodamine 110) were used for detection. All samples were filtered with a Millex-LG syringe filter (pore size 0.2 μm, Millipore) before analysis.

Evaluation of drugs encapsulated within nanoparticles by HPLC

Particle size measurement A Nanotrac Wave dynamic light scattering (DLS) instrument (Microtrac BEL Corp., Osaka, Japan) was used to measure the diameters of the nanoparticles. The measurement that was performed was the same procedure that was described in our previous paper [20]. Quantitation procedure Relative quantities of encapsulated drug were calculated as the ratio of the peak area of nanoparticles in the sample solution to the standard solution. The standard solution of the each measurement is described in the figure legends.

Result Encapsulated molecule type Amphotericin B (polyene antimycotic) and irinotecan (anticancer drug) were selected as typical encapsulated drugs [29]. Nanoparticles containing these drugs were prepared and their elution profiles were compared. Figure 1 shows chromatograms for the prepared solutions of amphotericin B-containing (24 nm) and irinotecan-containing nanoparticles (28 nm). Both nanoparticles eluted rapidly (about 7 min) and were well separated from the free drugs that were not encapsulated by the preparation reaction. By-product peaks were detected at 14 and 28 min for amphotericin B-containing nanoparticles (Fig. 1). A chromatogram of the rhodamine 110-containing nanoparticles (24 nm) is also shown in the insert of Fig. 1. The elution times for these nanoparticles were almost identical, suggesting that the nanoparticle elution time was not affected by the encapsulated molecules and that all

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nanoparticles were eluted mainly by the hydrodynamic mode. Although the significant peak tailing was observed for irinotecan-containing nanoparticle, the peak became symmetrical if the mobile phase was changed to a mixture of water and acetonitrile (data not shown). The relative standard deviation (RSD) values of the peak intensity for amphotericin Band irinotecan-containing nanoparticles were 6.7 and 1.6 %, respectively. These results indicated that this separation method can be applied to the analysis of various mixtures of nanomedicine and free drug. Calibration curve The relationship between the amount of encapsulated drug and peak intensity was examined by preparing standard curves. For selective detection of the encapsulated drug, UV detection at 410 nm for amphotericin B and fluorescence detection (excitation and emission at 365 and 440 nm, respectively) for irinotecan were used; detection was not hindered by absorbance or fluorescence of the silica nanoparticle. Because no certified reference material is available and there is a possibility that the spectroscopic property of the encapsulated drug is different from that of the free drug, the results are shown as relative values. Figure 2 shows the calibration curves of the nanoparticles; a good correlation was observed between the dilution and peak intensity. Thus, peak intensity changed in accordance with the amount of encapsulated drug. The determination coefficients (R2) of the calibration curves for amphotericin B- and irinotecan-containing nanoparticles were 0.981 and 0.999, respectively. We obtained good reproducibility in peak intensity (less than 5 % variation) and a strong correlation with the nanoparticle dilution ratio. Therefore, we concluded that the nanoparticles did not clog the column. The detection of a highly diluted sample (>100fold) was difficult in comparison to that of the free drug due to broadening of the nanoparticle peak. This broadening is caused by the differences in flow rate (flow rates for elution of nanoparticle and free drug were 0.2 and 0.5 mL/min, respectively) and the size distribution of the nanoparticle sample. The dynamic range of amphotericin B-containing nanoparticles (detected by UV) was narrower than that of irinotecan-containing nanoparticles (detected by fluorescence). Effect of the drug quantity in the preparation solution on the encapsulated amount of drug

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Time (min) Fig. 1 Chromatograms of the drug-containing nanoparticles and free drug. Magnified chromatograms of the nanoparticles are shown in the insert

Because good linearity was observed for the nanoparticle calibration curves, we evaluated drug encapsulation after preparation in varying drug concentrations. Three different batches of nanoparticles were prepared for two drugs (amphotericin B and irinotecan) and three quantities (0.05, 0.25, and 0.5 mg). The nanoparticle sizes and amounts of encapsulated drug are

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Fig. 2 Calibration curves of the drug-containing nanoparticles. Magnified calibration curve of the irinotecan-containing nanoparticles is shown in the insert

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summarized in Fig. 3; both qualities varied, even in nanoparticles prepared under the same conditions (standard errors for 0.25 mg amphotericin B and irinotecan were 0.046 and 0.76, respectively). The first step of the nanoparticle preparation is the hydrolysis of TEOS, which is the rate-determining step. Because the reaction occurs at the interface between the water and organic phases during mixing, it is difficult to control the reaction—unlike the relative simplicity of a homogeneous reaction. Batch-to-batch reproducibility in nanoparticle size and amount of encapsulated drug was better for amphotericin Bcontaining nanoparticles than for irinotecan-containing nanoparticles. We hypothesize that this difference is due to variations in the encapsulation amount (more irinotecan was encapsulated) and detection method (the fluorescence signal used for irinotecan detection is likely to be affected by the environment). The most homogeneous nanoparticles were prepared in a solution containing 0.25 mg amphotericin B. The amount of encapsulated drug prepared in different drug concentrations was compared using the average of

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three batches (Fig. 4). The encapsulated amount was increased by increasing the amount of drug in the preparation solution. Thus, the amount of encapsulated drug within the nanoparticles may be controlled by controlling the quantity of the drug in the preparation solution. However, it is essential to analyze the quantity of each nanoparticle preparation because the amount of encapsulated drug changes between batches. Therefore, this simple and fast analytical method is ideal for nanomedicine quality evaluations. The relationship between the amount of encapsulated drug and the quantity of the drug in solution differed between amphotericin B and irinotecan. When the quantity of the drug in the preparation solution was increased 5- and 10-fold, the encapsulated amphotericin B increased by only 1.5- and 2-fold; the increase in irinotecan was 3- and 7-fold, respectively. This difference is likely due to varying encapsulation efficiencies, perhaps driven by differences in drug hydrophobicity, which would reduce the encapsulation

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Fig. 3 The relationship between particle size and the amount of encapsulated drug in various preparations. The longitudinal axis is the value relative to the peak intensity of a nanoparticle prepared with 0.05 mg drug

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Drug quantity for nanoparticle preparation (mg) Fig. 4 The effect of drug concentration on encapsulation. The longitudinal axis is the value relative to the peak intensity of the nanoparticle prepared from 0.05 mg drug. *P

Rapid evaluation of the quantity of drugs encapsulated within nanoparticles by high-performance liquid chromatography in a monolithic silica column.

Drug-containing nanoparticles, the foundation of nanomedicine, provide promise for the safe and effective delivery of drugs to their targets. In this ...
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