The chemical structure of PEG5k
, a representative telodendrimer, is shown in . According to our nomenclature, “5k” represents the molecular weight of PEG (i.e. 5000 Dalton) and “8” indicates the number of CA subunits in the telodendrimer. PEG5k
is soluble in water and self-assembles into micelles with a size of 21 ± 4 nm. More importantly, it is capable of encapsulating hydrophobic drugs such as PTX. Bi-functional FmocNH-PEG-COOH is used as the starting material to construct the multifunctional telodendrimers (), and the azide group is introduced at the distal C-terminal of PEG and can be conveniently used for the ligation of cell surface targeting ligands (37
) or antibodies. The Boc protected amino groups adjacent to the dendritic core are reserved for the conjugation of fluorescent dyes, radionuclides, or drug molecules to the micelles, and hydrophobic drugs or probes can be physically encapsulated inside the micelles as shown in the cartoon in .
The chemical structure of PEG5k-CA8 with multiple functional groups and its size measured by DLS particle sizer, the cartoon illustrates the functionalized micelles formed by telodendrimers.
To study the physical properties of various telodendrimers and their effects on the in vivo
passive tumor targeting effects of the resulting nanoparticle, mono-functionalized MeO-PEG-NH2
with different PEG chain length (2–10 kDa) were used for linking various number of CA (4, 8 and 16) to the dendritic block to prepare a series of telodendrimers (). Representative structure of the inert telodendrimer is shown in Scheme S-1 in the supporting information
. The molecular weights of the telodendrimers were measured with MALDI-TOF mass spectrometry. The mono-dispersed mass traces were detected for the starting PEG and the telodendrimers. The molecular weight shifts between the telodendrimers and the corresponding starting PEG were almost identical to the mass increase by the conjugation of the oligo-cholic acids (Figure S-1 in supporting information
). The molecular weights of the telodendrimers from MALDI-TOF MS () were very similar to the theoretical value. The molecular weights of the telodendrimers were also determined by NMR spectrometry, based on the ratio of the proton signals of the three methyl groups on cholic acid (0.6–1.1 ppm) to the proton signals of the PEG (3.5–3.65 ppm) in the 1
H NMR spectra of the telodendrimers in CDCl3
(). The molecular weights obtained from NMR method were also very close to the theoretical values (), indicating the well-defined structures of telodendrimers. These telodendrimers were prepared via the well-established stepwise Fmoc peptide chemistry. The soluble PEG-supported conjugation reactions can be driven to completion by adding excess amount of reagents. Subsequently, the PEGylated intermediate and products can be easily isolated and purified via the precipitation and washing with cold ether, thus ensuring that the structures of the telodendrimers are well-defined. As shown , another advantage of this stepwise synthesis is that the structure of the telodendrimer can be easily engineered and multiple functional groups can be introduced into the polymer at particular density and at the specific sites.
The physical properties of telodendrimers with different PEG chain length and the number of cholic acids and lipophilic molecules.
Figure 4 (a) 1H NMR spectra of PEG5k-CA8 were recorded in CDCl3 and D2O, the suppression of cholic acid signals in D2O indicates the entanglement of cholanes in water by the formation of micelles; (b) CryoTEM images of PEG5k-CA8 loaded with PTX in the presence (more ...)
The critical micelle concentration (CMC) of the telodendrimers was measured with a standard fluorescence technique using pyrene as the probe.(36
) As shown in , the CMCs of the telodendrimers with different PEG chain length and the number of the cholic acid vary from 8 μg/mL to 250 μg/mL, corresponding to 0.97 μM to 67 μM (), due to the changes of the amphiphilicity. We found that the telodendrimers with larger number of cholic acids, such as PEG2k
, had low CMCs around 1 μM and yielded larger micelles with a heterogeneous size distribution. After loaded with PTX, significant precipitation was observed. Very low or undetectable PTX loading were observed after the removal of precipitates (). Micelles prepared from telodendrimers with medium CMC values (5–7.9 μM), such as PEG2k
, were found to be very stable and homogeneous in size before and after PTX loading. Those telodendrimers composed of fewer cholic acid molecules (PEG3k
) and with high CMCs (12.5 and 67 μM, respectively) were found to be able to form smaller micelles (10 and 15 nm, respectively) prior to drug loading. After PTX loading, the micelles increased in size up to 131 nm and 141 nm, respectively at relatively low PTX loading level, however, remaining very stable and homogeneous in size. While more PTX was added (>10% w/w drug/polymer), significant precipitation occurred for PEG5k
Figure 2 The CMC studies of the telodendrimers via fluorescence technique using pyrene as a probe molecule. The emissions of pyrene at 391 nm were collected through the excitation at 332 nm and 336 nm, respectively. The ratios of I336/I332 are plotted vs the concentration (more ...)
Most of telodendrimers discussed above, except PEG2k-CA8 and PEG10k-CA16, can be readily dissolved in water at concentration up to 200 mg/mL. In the micelle characterization, PTX loading and in vivo xenograft studies, we decided to keep the concentration of the telodendrimers at 20 mg/mL, which provided effective PTX concentration and exhibited the low viscosity needed for tail vein injection of the nanotherapeutics in the in vivo animal experiments. Through adjusting the PEG chain length and the number of CA subunits in the telodendrimers, the particle sizes of the PTX loaded micelles can be tuned ranging from 15 nm to 141 nm. Among them, PEG5k-CA8 has a medium particle size of 61 nm and the highest PTX loading capacity (7.3 mg PTX loaded in 20 mg PEG5k-CA8/mL), which is equivalent to 36.5% (w/w) of drug/polymer ratio (). The final particle size of PTX-loaded PEG5k-CA8 (with CMC around 5 μM) showed a modertate increase in size, while other telodendrimers, such as PEG3k-CA4 (CMC at 12.5 μM), produced a significant size increase at high drug loading level (). Our telodendrimer micelle systems can also effectively deliver other therapeutic agents including etoposide, a topoisomerase II inhibitor for cancer treatment, at an etopside/polymer ratio of 18.5% in PEG5k-CA8 (). By increasing the amount of drug added, the etoposide-loaded micelles exhibited very similar changes in sizes when combining with different telodendrimer micelles: PEG3k-CA8 and PEG5k-CA8 maintain their small particle sizes with high drug loading capacities, while and the size of etopside-PEG3k-CA4 micelles increases dramatically with the increased drug loading ().
Figure 3 The relationships of the particle size of the PTX and etoposide loaded micelles formed by different telodendrimers (a, c) and the drug/telodendrimer ratios in drug loaded micelles (b, d) vs. the feed ratios of drug/telodendrimers during the drug loading, (more ...)
We have also substituted cholic acid in PEG5k
with other natural lipophilic molecules such as cholesterol formate (CF), lithocholic acid (LA) (both with planar steroid scaffold), and heptadecanoic acid (HA) (linear fatty acid). The resulting telodendrimers, with low CMCs at approximately 1 μM, tend to form precipitate in aqueous solution and their PTX loading capacities are rather low (). The above studies indicate that cholic acid is essential to form stable telodendrimer micelles. Presumably, under aqueous environment, the facial amphiphilic cholic acids in the dendritic core can arrange their hydrophobic convex surfaces to the interior of micelle, and place the opposite hydrophilic surfaces against the aqueous environment to lower the energy, and together with PEG to stabilize the hydrophobic components inside the micelles. Additionally, some of the cholic acids might be randomly packed inside the hydrophobic core and their hydroxyl groups may form hydrogen bonds with the hydrophilic groups in PTX; such interactions may contribute to the good drug loading capacity of these micelles.(38
H NMR spectra of PEG5k
revealed the expected structure of the telodendrimer. However, when the 1
H NMR spectrum was collected in deuterated water, the cholane signals (0.6–2.2 ppm) were greatly suppressed (), indicating that the dendritic oligo-CA was tightly packed in the core of the micelles with high microviscosity. The similar phenomena were also reported in other micelles systems. (32
) In the cryoTEM study (), PTX was loaded into PEG5k
micelles (7.3 mg PTX/mL) in order to increase the density of the core and enhance the contrast against the background. Spherical micelles were observed with in situ
sizes ranging from 30 to 60 nm calibrated with tobacco mosaic virus (18 nm in width), which is consistent with the DLS measurement (61 ± 21 nm, ). Blank micelles (16 ± 4 nm by DLS) and PTX-loaded micelles (6.4 mg PTX/mL, 23 ± 8 nm by DLS) prepared from thiol-functionalized HS-PEG5k
(Scheme S-2 in the supporting information
) were immobilized on gold surfaces for atomic force microscopy (AFM) scanning. Tapping mode AFM topographs were obtained in aqueous solution. Both the empty and drug-loaded micelles appeared as individually immobilized nanoparticles with average sizes of 15 nm and 26 nm, respectively (), which were close to the particle sizes obtained by DLS measurement.
The morphology of the aggregates formed by other telodendrimers was studied by TEM. As mentioned above, PEG3k
when loaded with hydrophobic molecules such as PTX, tend to form large nanoparticles. Interestingly, rod shape aggregates (40 nm in width and several hundreds nm in length) were observed under TEM imaging of the PTX- PEG5k
sample (). PEG2k
, on the other hand, was observed to form gel or gel particles at high concentration (30 mg/mL) upon storage. After loaded with hydrophobic payloads, such as NIR dye (DiD) or PTX, the micelle solution became more viscous upon storage overnight at room temperature. Spherical micelles (15 nm) and nano-fibers (about 2 nm thickness) were observed under CryoTEM (). This indicates that the morphology could change from the spherical micelles (low viscosity) to fibers (high viscosity) during storage. The morphological outcome is probably determined by the structure as well as the amphiphilic balance of the telodendrimers. The packing parameters (ratio of hydrophobic/hydrophilic blocks) have been widely used to predict the morphology of the micelles formed by linear amphiphilic copolymers.(40
) However, the same rule may not apply to our telodendrimers with facial amphiphilicity and bulky cluster of cholic acids, Work is currently underway in our laboratory to study the relationship between the packing parameters and morphological outcome of the telodendrimer-based micelles.
Figure 5 (a) TEM images of the PTX loaded PEG5k-CA4 (20% w/w) solutions. The rod-like aggregates were observed with 40 nm in width and several hundreds nm in length (b) CryoTEM images of DiD loaded PEG2k-CA4 in the presence of tobacco mosaic virus (TMV) as calibration (more ...)
The stability of the PTX loaded PEG5k
micelles were followed by the DLS particle sizer. The particle size of these PTX-loaded micelles in aqueous solution was found to be highly stable at 4°C for over 6 months, no further aggregations and no needle crystals of PTX were observed. In contrast, most of the PTX-loaded polymeric micelles reported in the literature are lack of stability (from a few hours to days). (30
) Even Abraxane®, the FDA-approved albumin nanoparticle-bound PTX, tends to precipitate 24 hours after reconstituted with saline (Supporting information, Figure S-2
). Upon dilution with PBS to 125 fold to mimic the dilution by the blood pool through intravenous (iv) injection, needle-like crystals of PTX were observed in the diluted Taxol®
(Cremophor formulation of PTX) solution within a month. In contrast, this was not observed in the diluted PTX loaded PEG5k
micelle solutions even for 12 months, indicating that the PTX complex inside these micelles is very stable.
Upon dialysis in a cassette (MWCO 3500 Dalton) against PBS with different sink conditions over time, PTX can diffuse out from the micelle systems, but no burst release was observed (). Under the condition of frequently refreshed PBS during sampling, the drug release was much faster than that in the unchanged PBS. The condition with charcoal in the PBS reservoir (without changing PBS) allowed efficient absorption of the released PTX, which mimic the in vivo
protein binding and cellular uptake of drug, the drug release was even more faster than that in the refreshed PBS. Bovine serum albumin (BSA) was found to be not efficient to absorb the released free PTX, resulting in slow drug release rate, which is very similar to that in the unchanged PBS condition. In addition, PTX needle crystals were observed in the dialysate in the unchanged PBS and in BSA solution conditions, which lead to some variations in the measurements. However, the needle crystals were not found in the dialysates in the conditions of refreshed PBS and in the unchanged PBS with charcoal. Approximately 50% of the loaded PTX was released from the micelles after 24 hours in the sink conditions, and no burst release was observed during the fist few hours. Interestingly, we found that the PTX release rate of the samples, which had been prepared and stored for two months, were lower compared to the freshly loaded samples and the release pattern was in a linear mode (Supporting information, Figure S-3
). One possible explanation for this observation is the crystallization of PTX inside the core of the micelle or the crystallization of hydrophobic segments of the telodendrimer over time. To address this question, X-ray diffraction (XRD) studies were performed on these samples. Distinct PEG crystalline peaks were identified at 19.1° and 23.3° (42
) (Supporting information, Figure S-4
); however, no crystalline peaks for PTX (42
) or hydrophobic moieties (Cholic acid) were detected in the XRD spectra. Instead, the typical diffraction curve of the amorphous polymer was observed from 10° to 30°, indicating that the micellar core loaded with PTX was amorphous. The XRD studies of these old samples indicate the slow PTX release was not caused by the crystallization of PTX or the hydrophobic segments inside the micelles. The DSC (differential scanning colorimetry) analysis revealed an increased Tg (glass transition temperature, 200°C) of the cholane core for a PTX-PEG5k
sample lyophilized after storage for 55 days at 4°C, compared with the same sample lyophilized at earlier time point, such as day 5 (Tg: 179 °C) (Supporting information, Figure S-5
). Thus, the slow PTX release from the old PTX-loaded samples may be due to the more compact core structures formed by the rearrangement of the dendritic cholane segments during storage.
Figure 6 The accumulative paclitaxel released from micelles formed by PEG5k-CA8 in PBS (pH 7.4) at 37°C with different sink conditions: unchanged PBS; refreshed PBS, unchanged PBS with 10 mg/mL of BSA and charcoal, respectively. The concentrations of PTX (more ...)
The cytotoxicity of the telodendrimers in cell culture was evaluated via the MTT assays. As shown in , up to 1 mg/mL of PEG5kCA8 in the cell culture media, no significant cytotoxicity was observed on MDA-MB-231 breast cancer cells (IC50: 1.5 mg/mL). However, the telodendrimers with shorter PEG chain length (2 and 3 KDa) exhibited cytotoxic effects at relatively lower concentrations, and the IC50 of PEG2kCA4 PEG3kCA4 PEG3kCA8 were 45, 126 and 150 μg/mL, respectively. PEG5kCA8 remained to be rather non-toxic, compared to other telodendrimers with shorter PEG chains, to other cancer cell types including SKOV-3 (ovarian cancer) and HT29 (colon cancer). This may be explained by the extra stereo hindrance of the longer PEG preventing the interaction between the micelles and cells. The cell killing efficacies of PTX loaded telodendrimers, such as PEG5k-CA8, PEG3kCA4 and PEG3kCA8 and the two clinical formulations of PTX (Taxol® and Abraxane®) on SKOV-3 cells were found to be comparable, and the IC50 ranged from 4.3 to 6.2 ng/mL, after 72 hours of continuous incubation with the drugs (). The corresponding concentrations of the telodendrimers at the IC50 of PTX formulations are 20–30 ng/mL, which are far less than the IC50 of the blank telodendrimers (45–1500 μg/mL), indicating the potential clinical applications of these telodendrimers for delivery of PTX. The IC50 of PTX and PTX loaded PEG5kCA8 on the MDA-MB-231 cells, were observed to be higher than that in SKOV3 cells at 45 ng/mL and 17 ng/mL, respectively, over 72 hours of continuous drug exposure.
Figure 7 (a) The cytotoxicity of empty telodendrimer micelles against the MDA-MB-231 breast cancer cells; no significant toxicity for PEG5k-CA8 was observed at 1 mg/mL concentration, telodendrimers with short PEG chains show moderate toxicity at high concentrations (more ...)
To study the effects of particle size on the in vivo
biodistribution of nanoparticles, DiD (a hydrophobic near infra-red (NIR) cyanine dye, 1 mg/mL) was co-loaded with PTX (4 mg/mL, 4 mg/mL and 3 mg/mL, respectively) into 20 mg/mL micelle solutions of PEG2k
, to form three fluorescence labeled micelle preparations with distinct sizes of 17 nm, 64 nm and 154 nm, respectively (). Each of these three sizes of DiD-loaded micelles was incubated with Raw 264.7 macrophage cells for 2 h. The cells were then washed three times with PBS, fixed with 70% ethanol and observed under confocal fluorescence microscope. As shown in , the larger micelles (154 nm), compared to the two smaller ones, were preferentially taken up by the macrophages, which was subsequently confirmed by the flow cytometry analysis (data were not shown). NIR fluorescent imaging was used to evaluate the in vivo
biodistribution of the three micelles with different sizes in nude mice bearing the SKOV-3 ovarian cancer xenografts (subcutaneous implants). The mice were injected via the tail vein with the same volume of the above three PTX-DiD loaded micelle preparations in PBS. Ex vivo
imaging of the excised organs and tumors were performed at 24 h after injection. As shown in , the larger nanoparticles (154 nm) clearly exhibited the highest fluorescence intensity in the liver and the lungs, likely due to nonspecific uptake by macrophages in those organs; moreover, the fluorescent uptake by the tumor was very low. In contrast, tumor uptake of the smaller micelles (17 and 64 nm) was much higher than normal organs. The duplicated images in a different set of mice showed similar size effects on the biodistributions (Figure S-6 in supporting information
). The in vivo
NIR images of a tumor bearing mouse treated with the DiD-PTX-PEG5k
was recorded over time (). It is evident that the accumulation of the DiD loaded micelles at the tumor site via the EPR effect began at 2 h after injection, and continued to increase over the next 24 h period. In contrast, tumor targeting was not found in the tumor-bearing mice treated with the free dye.(33
) Consistent with imaging study of the size effect, PTX-loaded PEG5k
have been shown to be superior to Abraxane®
in the treatment of both subcutaneous and intraporitoneal xenograft ovarian cancer murine models.(33
) We believe PTX-PEG2k
, with smaller micelle sizes (17 nm), will be able to penetrate more deeply into the solid tumor and potentially result in better antitumor effects in tumor xenograft models. This study is currently undergoing in our laboratory.
Figure 8 (a) Three micelles, each with a different size after co-loaded with PTX and DiD, were measured with DLS particle sizer; (b) Raw 264.7 macrophage cells were incubated with each of the three micelle preparations, and then imaged under a confocal fluorescence (more ...)
In our previous report (33
), we were able to demonstrate that PTX-loaded PEG5k
(3 consecutive doses every 4 days) slowed down the growth of ovarian cancer xenograft (100–200 mm3
) considerably however, the tumors eventually progressed and complete cure was never achieved. In this study, we treated 5 groups of mice with 5 consecutive doses of drugs given i.v. every 4 days when the xeongraft implants reached 50 mm3
in size. As expected, rapid tumor growth was observed beginning on day 16 in the control group treated with PBS (). In contrast, no tumor growth was detected in mice treated with each of these five PTX regimens until day 35 when the tumors began to grow in the Taxol®
group at 15 mg/kg (MTD dosage for five consecutive treatment(44
)). By day 40, tumor growth also began in the Abraxane group at 30 mg/kg (MTD dosage for five consecutive treatment(44
)) and the low dose PEG5k
(15 mg/kg) group. For the high dose PEG5k
group at 45 mg/kg (MTD dosage for five consecutive treatment), the luciferase signal was undetectable by day 42 (supporting information Figure S-7
) and there was no signs of palpable tumor over 3 months, which indicated that there was no residual disease and complete cure was achieved. Unlike Taxol®
, myelosuppression from the PTX-loaded PEG5k
was minimal (supporting information Figure S-8
). No weight loss was observed in mice treated with these new nanoformulations (supporting information Figure S-9
), while consistent weight loss was observed in those treated with Taxol®
Figure 9 Anti-tumor efficacy of different PTX formulations in nude mice bearing human SKOV3-luc ovarian cancer xenograft. PBS (control), Taxol, Abraxane and the PTX-PEG5k-CA8 preparations were administered i.v. on days 0, 4, 8, 12, and 16 (marked with arrows) (more ...)
In summary, we have developed a well defined, engineerable and multifunctional telodendrimer system, which can self-assemble to form micelles in aqueous condition. By optimizing the structures of the telodendrimers, biocompatible nanocarriers have been obtained with tunable sizes and efficient PTX and etoposide loading properties. We have found that the size of the micelles can greatly influence their in vivo biodistribution. The smaller drug loaded micelles (17 nm and 64 nm) were more readily and efficiently to carry the loaded hydrophobic anticancer drugs to the tumor sites via EPR effects. In contrast, micelles with larger sizes (154 nm) were found to have very high uptake in liver and lung, but low uptake in tumor. In the in vivo antitumor efficacy studies in nude mice bearing early stage tumor xenografts, the toxicity profile and antitumor effects for PTX-loaded PEG5k-CA8 were observed to be superior to those of Abraxane® and Taxol®, and the cure of the disease was achieved in the group treated with PTX-loaded PEG5k-CA8 at its MTD dosage.