Micelles with Ultralow Critical Micelle Concentration as Carriers for Drug Delivery

Micelle-forming DSPE-PCB 5K.

Sharp-contrast micelle-forming molecule, DSPE-PCB 5K (a zwitterionic carboxy betaine polymer (PCB) of 5000 Dalton molecular weight (MW) conjugated to 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) lipid) was synthesized following our previous method,³⁵ and its structure is shown in Fig. 1 DSPE-PCB 5K powder is extremely water-soluble, and its resulting micelles were imaged by transmission electron microscopy (TEM) (Fig. 2a). The size of this micelle was measured by dynamic light scattering (DLS) (Fig. 2b), with a hydrodynamic diameter of 20±3.5 nm (mean ± SD, N=3) in phosphate buffered saline (PBS). The hydrophobic core domain of DSPE-PCB 5K micelle was detected using pyrene as reflected by the decrease in the intensity ratio of the first to the third highest energy bands (I₁/I₃) in the emission spectra of pyrene from 1.7 in water to 1.1 in micelle solution (Supplementary Fig. S1), following our previous method.³⁶ It should be noted that PCB such as 5K Dalton molecule weight is extremely water soluble without forming particles or aggregates (hydrodynamic diameter =3.7±1.0 nm). Aggregation Number (N_agg) of DSPE-PCB 5K micelle in water was 44, which was calculated from the ratio of an apparent molecular mass of micelle, measured via static light scattering³⁷, and the M_W for a single DSPE-PCB 5K chain.

Conventional methods cannot determine an ultra-low CMC.

CMC for DSPE-PCB 5K was determined to be 1.3 ×10⁻³ mM using the pyrene probe method (Supplementary Fig. S2).³⁶ The pyrene method is one of the most sensitive and precise methods of CMC determination.^38–40 However, it does not have sufficient sensitivity to measure a CMC below 10⁻³ mM⁴¹ because the final concentration for pyrene is maintained at 6 ×10⁻⁴ mM (in our previous protocol as well as others^{36, 42}) to give good quality fluorescent spectra. A micelle concentration approaching or below 10⁻⁴ mM cannot significantly change the aggregation behavior of pyrene, and potential micelle formation cannot be detected (as reflected by pyrene spectrum change). As a consequence, 1.3 ×10⁻³ mM as measured by the pyrene method can be an upper CMC estimation for DSPE-PCB 5K. There are other fluorescent dyes such as 2-p-toluidinylnaphthalene-6-sulfonate (TNS) and 1-anilinonaphthalene-8-sulfonate (ANS),^43–45 or other ways to determine CMC including light and X-ray scattering,^{46, 47} surface tension study,^{48, 49} and isothermal titration calorimetry.^{50, 51} None of them can precisely detect a CMC < 10⁻⁴ mM. Limited success in determining a CMC < 10⁻⁴ mM has been achieved by extrapolation of CMC values of structurally-similar molecules⁵² and through filtration of radioactively labeled surfactants.⁵³ However, these approaches are not readily available.

Verification of new method to determine CMC.

To explore a potential CMC below 10⁻⁶ M, we developed a “diluting-concentrating” method. An initial stable colloid was prepared by reducing soluble Au³⁺ ions to insoluble Au nanoparticles (NPs) in the presence of micelles at a concentration far above their CMCs. A very small Au NP (<5 nm) was formed as a non-soluble probe, partitioned into the hydrophobic core of a micelle stabilized. Au probe size was estimated from maximum absorbance peak of Au NP,⁵⁴ and also from the size increase from empty micelle to probe-loaded micelle (measured by DLS). The micelle-protected Au NPs were subject to serial dilutions in water, and extremely diluted micelle concentration can be reached. Potential aggregation of Au NPs resulted in an increase in both the hydrodynamic size of the colloid and the maximum absorbance wavelength for Au NPs,⁵⁴ as measured by DLS and UV-spectrometer, respectively. Consider the samples may be too diluted for these measurements, they were concentrated using a rotary evaporator at 35 °C under reduced pressure and then re-measured for these parameters. Potential aggregation of hydrophobic Au NP cannot be reversed by this concentrating process and was detected from their corresponding concentrated samples (Supplementary Text1). Since cargo aggregation is attributed to the dissociation of protecting micelles, this method indirectly detects the CMC values. A series of common micelles commercially available with known CMCs labeled by the manufacturers were used to verify this method, including non-ionic micelles (polysorbate 80, DSPE-PEG 5K and 2K), cationic micelles (hexadecyltrimethylammonium bromide (CTAB)), anionic micelles (sodium dodecyl sulfate (SDS)), zwitterionic carboxy betaine and sulfobetaine micelles with varied hydrophobic chain lengths (dodecyl dimethylglycine (CB12C), N-Tetradecyl-N-N-Dimethylglycine (CB14C), 3-(decyldimethylammonio) propane sulfonate (SB10C), 3-(N,N-Dimethylpalmitylammonio)propane sulfonate (SB16C)). Representative measuring curves are shown in Fig. 3. Measuring curves for all samples tested are shown in Supplementary Fig. S3. Without exception, CMC values for all micelles are right positioned, below which both the hydrodynamic size of the colloid and the maximum absorbance wavelength for Au NP start to increase significantly (Student’s t test, p<0.05). Similar to common probes for CMC determination (e.g., pyrene, dye molecules, etc.), the presence of Au NPs in the hydrophobic micelle core does not seem to influence the dissociation behavior of micelles or an artifact signifying that the colloid system does not aggregate at < CMC would appear.

DSPE-PCB 5K has an undetectable ultra-low CMC.

Using the validated “diluting-concentrating” method, DSPE-PCB 5K micelle protected Au NP does not show any signs of cargo aggregation at concentration down to 2.7×10⁻⁶ mM (Fig. 3; no significant signal increase based on student’s t test). Its CMC is considered ultra-low and below our current detection limit of 2.7×10⁻⁶ mM. The detection limit can be potentially lowered by one order of magnitude by further diluting the micelles, e.g., into a 10 L container, and then concentrating 1–1.5 L each time (using the currect 2 L evaporation flask); this approach is laborsome and has little room to significantly lower the CMC detection limit. In contrast, DSPE-PEG 5K micelles, widely used to construct nanomedicines, have an apparent CMC about 1×10⁻³ mM, consistent with literature values^{41, 55, 56}, and dissociate at concentrations, at least, three orders of magnitude higher than DSPE-PCB 5K. Compared with DSPE-PCB 5K having multiple zwitterionic carboxy betaines as the polar group, CB14C contains only one zwitterionic carboxy betaine and has significantly higher CMC at 3.4×10⁻² mM. To confirm that DSPE-PCB 5K truly has an ultra-low CMC, we performed the following experiments: 1) Au NP colloid was formed in the presence of PCB 5K replacing DSPE-PCB 5K and was found unstable (aggregate). Note that DSPE-PCB 5K/Au colloid was stable at room temperature for weeks without precipitation. This excludes the possibility of PCB tightly interacting with Au NPs, preventing dissociation of DSPE-PCB 5K micelles (an artifact of lowered CMC). 2) Au NP probe was replaced by cross-linked polystyrene (PS) NP probe. Following the same “diluting-concentrating” method, where colloid aggregation was detected by DLS, CMC results were obtained similarly to Fig. 3a, regardless of the probe used (Supplementary Fig. S4). It should be noted that both Au and crosslinked PS NPs are non-dissolvable under extremely diluted conditions. This is the key to obtaining reliable CMC measuring results. Dissolvable salt NPs (e.g., CaCO₃) were not found to work as a probe since they are released from the micelle at a diluted concentration below their water solubility and aggregated when concentrated to the concentration above their water solubility (Supplementary Fig. S5). 3) DSPE-PCB 5K micelle at extremely low concentration (2.7×10⁻⁶ mM) has been further observed using TEM (Supplementary Fig S6).

Zwitterionic PCB and its amount are critical to achieving ultra-low CMC property.

To explore the unique role of zwitterionic PCB contributing to ultra-low CMC property, we incubated either free form of CB moiety or PCB 5K (with multiple CB) at high concentration with conventional micelles (i.e., SDS and Polysorbate 80), and found that conventional micelles were significantly stabilized in both cases (Fig. 4a, b, and Supplementary Fig. S7a, b). It should be noted that similar stabilizing effect has been found in both inorganic and organic salt solutions.^{57, 58} We attribute this effect to the super-hydrophilicity of CB and PCB (similar to salts), which increases the polarity of the aqueous phase,^{35, 59} strengthens the hydrophobic-hydrophobic interaction, and drives the micelle assembly at a concentration far below the micelle’s inherent CMC. The presence of ethylene glycol oligomer (OEG) or polymer (PEG), in contrast, increased the CMC for conventional micelles (Fig. 4a, b, and Supplementary Fig. S7a, b), which is consistent with the literature.⁶⁰ OEG and PEG are known for their hydrophobic nature and tend to decrease the polarity of the aqueous environment,^{35, 59} behaving the opposite to super hydrophilic CB and PCB (Supplementary Text2). We further examined the micelle stabilizing effect of CB or PCB 5K at a series of concentrations, and found that a minimum of roughly 200 mg/ml CB and 40 mg/ml PCB 5K were required to prevent the dissociation of conventional micelles, e.g., SDS and Polysorbate 80 (Supplementary Fig. S7c, d, e, f). Thus, the presence of zwitterionic CB moiety as polar groups for DSPE-PCB 5K appears to be the key to the ultra-low CMC property.

We further found that an appropriate molecular weight (MW) of PCB (amount of CB moieties per polymer) is critical for DSPE-PCB to achieve the lowest possible CMC (Fig. 4c, d). PCB MW is expected to be as high as possible to ensure the CMC lowering effect. However, PCB MW shouldn’t be too large since micelles with longer hydrophilic polymer chain typically have an increased CMC (e.g., shown by hydrophilic polyvinylpyrrolidone (PVP)-hydrophobic alkyl chain micelles⁴¹). It appears that DSPE-PCB 5K has so far, the balanced PCB MW and achieves the lowest possible CMC < 10⁻⁶ mM (undetectable), comparing with DSPE-PCB 2K (CMC= 1.2 – 6 ×10⁻⁵ mM) and 10K (CMC= 0.9 – 1.8 ×10⁻⁵ mM), and other reported micelles containing zwitterionic polymer block.⁶¹

Ultra-stability of ultra-low-CMC Micelle when diluted in serum.

One major challenge with drug delivery is the dissociation of carrier entities (e.g., micelles) as they are infused into the blood stream since the drug carrier is typically diluted below its CMC. Blood serum comprises a large number of proteins (albumin) that decompose micelles and bind with this surfactants.⁶² Compared with water, dilution in serum is even more challenging for assembled carrier systems. Here we tested the stability of ultra-low CMC and conventional micelles in fetal bovine serum (FBS) using the Au NP probe at 0.055 mM micelle concentration (Fig. 5). We found that DSPE-PCB 5K (ultra-low CMC) showed excellent stability over 72 h. Conventional Polysorbate 80 and DSPE-PEG 5K micelles were not stable; for DSPE-PEG 5K, it dissociated at this concentration even above its CMC. The diluted micelle concentration tested (10⁻² mM) is chosen by considering the situation of a 60 kg adult patient having 4.5 L blood receiving 75 mg/m² TAXOTERE® to treat breast cancer.⁶³ The hypothetical maximum micelle concentration (i.e. Polysorbate 80) in the blood is calculated to be 5.5 × 10⁻² mM, assuming all drug was injected simultaneously. The reality is TAXOTERE® has to mix with infusion saline and be slowly infused (typically complete in one hour).⁶⁴ As drugs gradually enter the blood, they are immediately diluted, with micelle concentration significantly lower than 10⁻² mM. Our serum stability test at 10⁻² mM indicates that ultra-low-CMC micelle is far more stable than conventional ones as drug carriers at clinic-relevant concentrations.

Drastically increased antitumor performance of docetaxel stabilized by ultra-low-CMC micelle.

To study the applicability of using ultra-low CMC micelles to deliver real drugs, we chose docetaxel and prepared docetaxel/ DSPE-PCB 5K formulation using a simple thin-film hydration method (see Experimental Section)^65–68. Our controls include docetaxel/Polysorbate 80 and docetaxel/DSPE-PEG 5K, similarly made through thin-film hydration method, and TAXOTERE® formulated based on literature^{63, 64, 69}. The weight ratios of drug over surfactant for all formulations were kept at 4%, which is the drug loading level for TAXOTERE®^{66, 68}. The hydrodynamic diameters for all tested samples were around 22 nm as determined by DLS. However, after diluting these samples in FBS to reach micelle concentration of 5.5 X10⁻² mM (the hypothetical maximum micelle concentration in the blood), only docetaxel/ DSPE-PCB 5K can maintain its hydrodynamic size, while all other control formulations appeared to be un-stable over the tested time period (Fig. 6a). Drug release profiles were determined by placing the formulations into Slide-A-Lyzer MINI dialysis microtubes (3500Da MW cutoff) and dialyzed against PBS at 37°C with gentle stirring. Docetaxel remained inside the dialysis tubes at different time points was quantified using HPLC to calculate drug release. DSPE-PCB 5K showed the highest retention of docetaxel compared with other control micelles, with 50% drug released in 3.7 h (Supplementary Fig.S8). The drug release mechanism in our formulation is expected to be based on drug dissolution or drug leaching. This has been indicated by our control study showing the zwitterionic micelles can stabilize non-dissolvable Au NPs and crosslinked PS NPs (Fig.5, Supplementary Fig. S4) but not dissolvable CaCO₃ NPs (Supplementary Fig. S5) at diluted concentrations. We further checked the stability of different docetaxel formulations after lyophilization. Results indicated that after lyophilization docetaxel/DSPE-PCB 5K maintained its original hydrodynamic size, while all control micelle formulations significantly aggregated (Supplementary Fig. S9).

The maximum tolerated dose (MTD) of docetaxel/ DSPE-PCB 5K was determined using healthy C57BL/6 mice to be 55 mg docetaxel/kg bodyweight, significantly higher than control formulations docetaxel/DSPE-PEG 5K (40 mg/kg), docetaxel/Polysorbate 80 (40 mg/kg), and TAXOTERE® (25 mg/kg) (Supplementary Fig. S10). To examine the anti-tumor activity of different docetaxel/micelle formulations, C57BL/6 mice were inoculated with B16F10 cells at right flank and on day 8 after inoculation received intravenous injection of drug formulations. The dose for all drug/micelle formulations was kept the same at 10 mg docetaxel/ kg body weight for 5 injections every other day. It was found that docetaxel/DSPE-PCB 5K showed significantly better anti-tumor activity compared with all other micellar docetaxel formulations, including TAXOTERE® (Fig. 6b). All tumors treated with this formulation were eliminated by 33 days after the first treatment without recurrence over 70-day observation period after tumor innoculation (Fig. 6c, d). None of the control formulations based on docetaxel could fully inhibit the melanoma tumor, and in fact other chemo drugs have rarely shown a complete elimination to melanoma^{28, 30, 32, 70–74}.

To further investigate the in vivo circulation profile of docetaxel/DSPE-PCB 5K, different docetaxel formulations were injected intravenously to healthy C57/BL6 mice. At different time points blood samples were collected and the concentration of docetaxel in plasma was measured via HPLC. It was found that docetaxel/DSPE-PCB 5K showed significantly longer blood circulation than all other micellar docetaxel formulations (Fig. 6e). The α and β half-life of docetaxel/DSPE-PCB 5K were calculated to be 0.6 hour and 5.9 hour, while for TAXOTERE® these values were 4 min and 36 min, respectively. For bio-distribution, healthy C57/BL6 mice were treated with intravenously injection of different docetaxel formulations after inoculation of B16F10 cells. Bio-distribution of docetaxel at different time points has been further studied. Significantly higher tumor accumulation of docetaxel/DSPE-PCB 5K was observed at 24 hours post injection compared with control formulations (Fig. 6f). Such enhanced drug accumulation to tumor site has been observed at as early as 2 hours after administration (Fig. S11). We attributed the remarkable antitumor performance of docetaxel in our formulation to the unprecedented stabilization ability by the ultra-low CMC micelles.

In summary, the Ultra-low CMC micelle will be applicable as a novel agent carrier based on its ultra-stability in protecting cargo (without dissociation) at extremely diluted conditions, including serum dilution at clinically relevant settings where conventional micelle systems fail. It is reasonable to predict that by replacing conventional micelles with the ultra-low CMC micelle, therapeutic outcome of the drug delivered shall be significantly promoted. Our data fully support this claim and show that the docetaxel/ ultra-low CMC micelle formulation can completely eliminate the melanoma tumor on mice, while most known chemotherapy, including TAXOTERE® was unable to do so. We expect the ultra-low CMC micelle can be utilized to enhance the delivery and therapeutic effects of other drugs in general.

Materials

DSPE-PCB 2K, 5K, and 10K were synthesized and characterized following the previously established method.³⁵ The molecular weight of NHS-PCB was characterized via Waters Breeze 2 system gel permeation chromatography (GPC) with Waters 2414 reflex detector. The mobile phase was phosphate buffered saline (PBS) at a flow rate of 1mL/min. Polysorbate 80, SDS, CTAB, HAuCl4, NaBH₄, styrene, divinylbenzene, trifluoroethanol were obtained from Sigma-Aldrich. (St. Louis, USA). DSPE-PEG 5K was obtained from Laysan Bio, Inc. (Arab, USA). CB12C, CB14C, SB10C. SB16C were obtained from Anatrace Product. LLC (Maumee, USA). Docetaxel was obtained from LC Laboratories (Woburn, USA). B16F10 cells were obtained from ATCC.

Preparation of Au NPs encapsulated by DSPE-PCB 5K:

CMC for DSPE-PCB 5K was determined via established pyrene method.³⁶ During a typical “Diluting-concentrating” method for CMC determination, a probe (<5 nm) is first loaded into micelle core at high micelle concentration (> CMC). To load Au NP probe, micelles and HAuCl₄ are dissolved in 10 ml deionized water and vigorously stirred at room temperature, followed by dropwise addition of 1 ml NaBH₄ aqueous solution (micelle: Au: NaBH₄ (molar ratio) is typically 1: 1: 6; take CTAB for example, its concentration is 0.01 M > its CMC 0.92 mM). The solution turns red, and the stirring is kept for another 5 min. To load cross-linked PS NP probe, micelles, photo-initiator 2959, styrene and divinyl benzene (molar ratio is typically 1: 10: 1.5: 1; take DSPE-PCB 5K for example, its concentration is 0.3 mM) are sonicated in deionized water to form nano-emulsion, followed by 20 min reaction under UV light. Micelles with either Au NP or PS NP loaded are further purified through 10K MWCO ultrafiltration to remove rough materials forming the probes, and 0.2 μm filter to remove unexpected large particles. The size of the probes can be determined by DLS (Zetasizer Nano-ZS, Malvern Instruments) by taking the size difference between empty micelles and loaded micelles. For Au NP probe, its size can be additionally measured by the maximum absorbance wavelength using a UV-VIS spectrometer (Thermo Scientific Multiscan Go). During the diluting-contributing procedure for CMC determination, the liquid sample was not further treated with filtration.

Aggregation Number (N_agg) of DSPE-PCB 5K micelle was calculated from the ratio of an apparent molecular mass of micelle which was measured via static light scattering (SLS) and M_w for a single DSPE-PCB 5K chain. Apparent molecular mass of DSPE-PCB 5K micelle was obtained by measuring a series of samples with different concentrations (ranged from 1.2 g/L to 4.8 mg/L) via Malvern Zetasizer Nano-ZS.

Preparation of docetaxel/DSPE-PCB 5K formulation

To prepare docetaxel/DSPE-PCB 5K formulation, 150 mg of DSPE-PCB 5K and 6 mg docetaxel were co-dissolved in 5 ml trifluoroethanol in a round-bottom flask. The solvent was subsequently evaporated by rotary evaporation to obtain a thin film. The film was then kept in a vacuum for 2 hours at room temperature to remove the residual trifluoroethanol. After the addition of 3 ml saline and stirring at 800 rpm for 30 min, a clear micelle solution with docetaxel loaded was obtained. Docetaxel/DSPE-PEG 5K and docetaxel/Polysorbate 80 formulations were prepared following the same procedure. Taxotere was prepared follow the reference^{63, 64, 69}. The weight ratios of drug over surfactant for all formulations were kept at 4%, which is the drug loading level for TAXOTERE®^{66, 68}.

Drug release profiles were determined by placing 100 μL different formulations into Slide-A-Lyzer MINI dialysis microtubes (3500 Da MW cutoff) and dialyzed against PBS at 37°C with gentle stirring. At different time intervals, docetaxel remained in the dialyzer was quantified through HPLC by a Waters Breeze 2 System equipped with a Waters 2998 photodiode array detector at 230 nm and a Waters symmetry C₁₈ column (4.6×7.5 mm). The mobile phase was a mixture of acetonitrile/water (50%:50% volume) at a flow rate of 1 mL/min. The docetaxel showed a single elution peak at 5.47 min.

Animal experiments

Animal experiment was approved by the Institutional Animal Care and Use Committee (IACUC) of Wayne State University, and performed in compliance with the relevant laws and institutional guidelines. B16F10 cells (ATCC, CRL-6475) were culture in RPMI 1640 medium containing 10% fetal bovine serum at 37 °C under 5% CO₂ atmosphere. For in vivo tumor therapeutic study, healthy C57BL/6 mice (7 weeks old, 20–25 g) received a subcutaneous injection of 100 μL B16F10 cells (5×10⁶ cells/mL) at the right flank. After 8 days of inoculation, the tumor became visible, and different formulations were administered by intravenous injection via tail vein 5 times every other day at the dosage of 10 mg docetaxel/kg body weight. The tumor volume of the mice was measured day by day and calculated with an equation of (L×W²)/2 where L is the long length and W is the short length of the tumor. Mice were euthanized if their weight loss exceeded 15% or if their tumor grew to a volume greater than 1000 mm³. For MTD study, healthy C57BL/6 mice received one intravenous injection of different docetaxel formulations through the tail vein at the dose of 25 mg/kg, 40 mg/kg, and 55 mg/kg. Treated mice were subsequently observed day by day and checked for mortality, body weight and signs of toxicity. To test docetaxel concentration in plasma, healthy C57BL/6 mice (7 weeks old, 20–25g) were treated with intravenous injection via tail vein at the dosage of 10 mg/kg. At different time intervals, 50 μL blood samples were collected and transferred to sodium heparin treated tubes. Docetaxel was extracted from the plasma supernatant by ethyl acetate and the extracted solution was evaporated under a gentle stream of nitrogen. The extraction residues were reconstituted in the mixture of acetonitrile/water (50%:50% volume). The docetaxel concentration was measured using HPLC as mentioned above.

To test bio-distribution, healthy C57BL/6 mice (7 weeks old, 20–25g) received a subcutaneous injection of 100 μL B16F10 cells (5×10⁶ cells/mL) at the right flank. After 8 days of inoculation when tumors became visible, different formulations were administered by intravenous injection via tail vein. Mice were euthanized at certain time point. Organs such as liver, heart, kidney, spleen, lung and tumor tissues were collected, thoroughly washed with saline (0.9% NaCl), weighted and stored for further analysis. Organs or tissues were homogenized with Mini-beadbeater-24 (Biospec Products, Inc., USA) in acetonitrile/water (50%:50% volume). The resulting 200 μL samples of homogenized tissue were mixed with 1 mL ethyl acetate. The samples were extracted on a vortex mixer for 2 minutes then centrifuged at 4000 g for 5 minutes. The organic layer was transferred to a clean tube and evaporated under a gentle stream of nitrogen. The extraction residue was reconstituted in 1 mL mixture of acetonitrile/water (50%:50% volume). The docetaxel concentration was measured using HPLC.

Statistical Analysis

n = 7 was used for mice per treatment group unless specified otherwise. The sample size was selected according to previous reports. All tested mice were included in analyses except for ones with unforeseen sickness or morbidity. Animal cohorts where randomly selected and investigator were not blind to performed experiments. Most data were normally distributed and similar variance was determined for groups that are compared. Analysis includes unpaired two-tailed t-test and one-way analysis of variance (ANOVA) with Bonferroni multiple-comparison correction. For data not following normal distribution, non-parametric Mann-Whitney test was used. *: P < 0.01, **: P < 0.001, ***: P < 0.0001 and n.s.: no significant difference. For all statistical analysis, significance was accepted at the 95% confidence level, and all analyses were two-tailed.