Reassessment of long circulation via monitoring of integral polymeric nanoparticles justifies a more accurate understanding

Haisheng He a, Sifan Jiang b, Yunchang Xie a, Yi Lu a, Jianping Qi a, Xiaochun Dong a, Weili Zhao ac, Zongning Yin b and Wei Wu *a
aKey Laboratory of Smart Drug Delivery of MOE and PLA, School of Pharmacy, Fudan University, Shanghai 201203, China. E-mail:
bWest China School of Pharmacy, Sichuan University, Chengdu 610041, China
cKey Laboratory for Special Functional Materials of the Ministry of Education, Henan University, Kaifeng 475001, China

Received 19th January 2018 , Accepted 21st March 2018

First published on 22nd March 2018

Monitoring of payloads results in a biased perception of long circulation of nanoparticles. Instead, herein, the long-circulation effect was re-confirmed by monitoring integral nanoparticles, but circulation was not found to be as long as generally perceived. In contrast, disparate pharmacokinetics were obtained by monitoring a model drug, paclitaxel, highlighting the bias of the conventional protocol.

Conceptual insights

Long circulation has become a paradigm in nanoscale drug delivery, especially for chemotherapy via intravenous administration. However, the long-hold perception of long circulation is flawed because the conceptual establishment is heavily based on monitoring of a surrogate such as a drug or a label. However, this study reassesses the phenomenon of long circulation via monitoring of integral nanoparticles. Our findings justify a more accurate understanding of long circulation. The “L”-type blood pharmacokinetic profiles demonstrate far less “longer” circulation time than usually perceived. Even densely PEGylated nanoparticles are cleared from the blood stream rapidly following intravenous administration and concomitantly accumulate in RES organs such as liver, spleen and lung. However, the long circulation effect is re-confirmed, but is only prominent within the beginning 8–24 h, depending on the degree of PEGylation. Less PEGylated and non-PEGylated nanoparticles are sequestrated almost completely by RES organs, with a small fraction re-discharged into circulation later. The long-circulation effect also results in enhanced distribution into peripheral tissues. These new insights are of significance in justifying a more accurate understanding of long circulation and provoke rational exploitation of this updated concept.

Intravenously administered nanoparticles, commonly dozens to hundreds of nanometres large, are primarily taken up by macrophages via phagocytosis in organs, such as liver, spleen, lung, and kidney, or tissues of the reticuloendothelial systems (RES); this is accompanied by their fast clearance from the blood stream.1,2 To achieve long circulation and subsequently divert nanoparticles to non-RES tissues, it is common to decorate nanoparticle surfaces with long-chain hydrophilic biomaterials such as polyethylene glycols (PEGs) and poloxamers.3–7 Long-circulation or stealthy decoration is believed to camouflage the nanoparticles such that to evade biorecognition and achieve longer circulation in the blood stream.2–4 The concept of long circulation further evolves to be the footstone of the enhanced permeability and retention (EPR) effect in tumours and inflammatory tissues.8–10 Over the years, the concept of long circulation, as well as EPR, has been repeatedly proven by experimental observations. However, the common perception of long circulation is flawed because of one long-ignored fact—the conceptual establishment is primarily based on monitoring of payloads such as a model drug or a radio/fluorescent label.8–15 It is arguable that the payloads may leak during their voyage in the body, a process that is doomed to happen with regard to biodegradable nanoparticles.16 In fact, previous findings merely reveal the behaviours of the payloads, a mixture of both encapsulated and released, and cannot be speculated to extrapolate the behaviours of the nanoparticles per se. This appears to be a potential reason why different results are obtained for the same nanovehicles,11–13 even in the same lab,14,15 using different model drugs. We hypothesize that the understanding of long circulation should be more accurate if appropriate approaches are employed to monitor integral nanoparticles rather than payloads.

Recently, our group developed a novel strategy to discriminate nanoparticle-associated signals from those of free probes in vivo.17,18 This strategy takes advantage of the water-quenching properties, commonly termed as aggregation-caused quenching (ACQ) of a new kind of environment-responsive near-infrared fluorescent probes used to label the nanoparticles.17,18 The ACQ effect is defined as the stacking of planar fluorescent probes in an unfavourable environment, e.g. hydrophobic entities in water, that results in spontaneous quenching of fluorescence.19 ACQ is normally regarded as an adverse effect and should be avoided by all means for efficient bioimaging. On the contrary, we exploited this property to achieve accurate monitoring of nanoparticles in vivo or in biological tissues by live imaging. These kinds of probes have a basic BODIPY or aza-BODIPY parent structure and near infrared emission, high quantum yield, high chemical stability, and safety. When molecularly dispersed in a favourable environment, e.g. in the matrix of nanoparticles, the probes are activated and emit fluorescence, but the fluorescence is quenched spontaneously and absolutely when they are released in an unfavourable environment, e.g. an ambient aqueous medium, due to the ACQ effect. Based on this ideology, we managed to investigate into the in vivo fate of a series of nanoparticles.11,17,18,20,21 All conclusions obtained from related studies, including this study, are based on the assumption that the captured fluorescence signals represent integral nanoparticles. It is believed that the interference owing to free probes can be reduced to as small as possible; this is a distinctive feature that makes these kinds of probes stand out from conventional non-environment-responsive probes.

Previous studies indicates that the stealthy coating should be as tight and dense as possible to efficiently evade opsonisation by various opsonins. Therefore, the quality of the long-circulating coating is very important. To perform a meaningful comparison with past findings, we have used copolymers with a methoxy PEG (mPEG) segment of 5k, which has been commonly used in previous studies22–24 and believed to provide the best camouflaging efficiency,25 to prepare PEGylated nanoparticles. Various previous studies have established a positive correlation between PEG chain length, or coating density, and long-circulation efficiency, albeit by monitoring of model drugs.26–31 In this study, we reassessed the long-circulation phenomenon as a function of the stealthy coating chain length and density via monitoring integral nanoparticles to validate or rectify the general understanding of long circulation. Moreover, the results obtained were compared with the behaviours of similar nanoparticles by loading a model drug, paclitaxel (PTX), as well as the behaviours of the payload drug itself. Although the drug fate is of more therapeutic relevance, elucidation of the fate of the nanoparticles may be of more toxicological significance and may help to elucidate the underlying mechanisms.

The model PEGylated nanoparticles, without loading paclitaxel, were prepared by an emulsification/solvent evaporation method using polycaprolactone (PCL) as the matrix and mPEG-PCL as the decorating material. Table S1 (ESI) summarizes the formulations and fundamental properties, such as particle size, polydispersity index (PDI), zeta potential, fluorescence intensity, and weight percentage of mPEG-PCL in the nanoparticle matrix, of the various mPEG-PCL nanoparticles studied herein. Surface coating was adjusted by either using mPEG-PCL of different mPEG chain lengths (5k vs. 2k) or incorporating different proportions (29%, 17% vs. 9%, w/w) of mPEG-PCL into the polymer matrix to achieve different coating densities, and non-PEGylated PCL nanoparticles were used as a reference. To investigate the particle size effect, both 200 nm and 80 nm nanoparticles with different coating densities (29% and 9%, respectively) were prepared. Transmission electron microscopy (TEM) reveals spherical morphology, very good shape, high dispersibility, and uniform size distribution (Fig. S1, ESI). Measurement by a nanosizer confirms comparable particle sizes among the same size category (200 nm or 80 nm) and quite narrow size distribution as indicated by a relatively small PDI. The zeta potentials are slightly negative for all formulations, which are in accordance with previous findings on PEGylated nanoparticles.32,33 Since the zeta potentials are nearly neutral, we have excluded this variable in downstream investigations. Moreover, to perform a parallel comparison between groups, the fluorescence intensity of all formulations was adjusted to similar levels.

The steadiness of fluorescence intensity is a crucial indicator that ensures the detection accuracy. For instance, if the fluorescence intensity decreases in aqueous buffers, there seems to be possibility of unwanted soaking of the polymer matrix or diffusive release of the probes into the surrounding medium that would interfere with the measurement.11,17 Results indicate that both the particle size and fluorescence intensity of all the formulations remain stable for at least 48 h in a pH 7.4 phosphate buffered saline (PBS) (Fig. S2A and B, ESI). The maintenance of steady particle sizes is a strong indicator of the well-dispersed state. The fluorescence intensity of the nanoparticles in the buffers remains steady (100 ± 10%) over a period of 48 h. However, the kinetics in the plasma is different with total fluorescence intensity decreasing gradually to approximately 85% and 70% at 24 and 48 h, respectively (Fig. S2C, ESI), due to slight degradation of the nanoparticles in the plasma. The diminishment of fluorescence reflects good mimicking of the virtual situation, rather than a compromise of the detection accuracy, following intravenous administration.

Following intravenous injection, the pharmacokinetics of mPEG-PCL nanoparticles was monitored by measuring blood-borne fluorescence. According to Fig. 1A, it is obvious that the overall elimination rate from blood is very fast with typical “L”-type biphasic kinetics, irrespective of the degree of PEGylation. Non-PEGylated PCL nanoparticles are rapidly cleared from the blood stream, with only 4.26% remaining after 15 min; this indicates probable phagocytic uptake by RES tissues as a result of spontaneous opsonisation by blood complementary systems.34–36 For all PEGylated groups, the blood fluorescence levels drop to less than 55% and 20% at 30 min and 4 h, respectively. Despite this, the long-circulation effect owing to PEGylation is apparent, as highlighted by the comparatively higher levels of nanoparticles in blood for a time duration of about 8–24 h depending on the degree of PEGylation. Using the AUC values as a measure, the effect of various variables on long circulation can be compared (Table 1). A positive correlation between the circulation time and PEG density is established in the following order: 29% ≈ 17% ≫ 9% ≈ 0% (Fig. 1A and D), with AUC0-t values 2.62, 2.02, and 1.49 times those of non-PEGylated nanoparticles (Fig. 1G). In Fig. 1B and E, a comparison between PEG chain lengths highlights the superiority of longer mPEG chains with a doubled AUC0-t value (5k vs. 2k) (Fig. 1H). A reduction in the particle size from 200 nm to 80 nm significantly prolongs the overall circulation time (Fig. 1C and F), especially at a lower PEG coating density of 9% with a 2.07-fold increase in AUC, contrary to only a 1.39-fold increase for the 29% group (Fig. 1I). Furthermore, we should pay attention to another apparent phenomenon in the pharmacokinetics of both non-PEGylated and less PEGylated nanoparticles that there is resurgence in the levels of blood-borne nanoparticles after the initial quick clearance, beginning at 1 h and peaking at around 8 h.

image file: c8nh00010g-f1.tif
Fig. 1 Blood pharmacokinetic profiles of mPEG-PCL and PCL nanoparticles in rats (n = 3) as highlighted by comparing PEGylation density (A), PEG chain length (B) and particle size (C), respectively. Plot D, E & F are enlargement of the 0–8 h regions of plot A, B and C, respectively, while bar plot G, H & I present corresponding AUC values (n = 3) as summarized in Table 1. The fluorescence intensity, indicated by total radiant efficiency (TRE), is normalized by setting the fluorescence intensity of the first sampling at 15 s as 100%. The fluorescence intensity stands for the relative quantity or mass of the nanoparticles.
Table 1 Pharmacokinetic parameters of nanoparticles obtained by fitting to a two-compartmental model
mPEG5k-29%-80 nm mPEG5k-9%-80 nm mPEG5k-29%-200 nm mPEG5k-17%-200 nm mPEG5k-9%-200 nm mPEG2k-29%-200 nm PCL-200 nm
t 1/2α (h) 0.79 ± 0.24 0.59 ± 0.16 0.78 ± 0.27 0.46 ± 0.23 0.13 ± 0.01 0.20 ± 0.13 0.07 ± 0.04
t 1/2β (h) 14.56 ± 2.1 17.27 ± 1.55 16.52 ± 6.73 17.11 ± 11.68 69.32 ± 0.00 34.67 ± 19.26 56.23 ± 22.66
K 10 (L h−1) 0.20 ± 0.04 0.22 ± 0.01 0.25 ± 0.07 0.36 ± 0.1 0.59 ± 0.35 0.70 ± 0.42 2.33 ± 1.63
K 12 (L h−1) 0.62 ± 0.44 0.91 ± 0.42 0.64 ± 0.54 1.42 ± 1.28 4.83 ± 0.25 3.49 ± 1.72 8.67 ± 6.36
K 21 (L h−1) 0.22 ± 0.07 0.22 ± 0.07 0.17 ± 0.06 0.26 ± 0.17 0.1 ± 0.01 0.16 ± 0.01 0.13 ± 0.13
AUC0-t (%*h) 418.39 ± 60.72 356.52 ± 17.98 301.56 ± 42.09 232.38 ± 42.11 172.23 ± 13.76 152.74 ± 27.80 115.23 ± 36.81
MRT (h) 12.08 ± 0.47 13.38 ± 0.83 11.58 ± 3.07 12.68 ± 0.95 19.59 ± 1.40 14.66 ± 1.15 19.081 ± 3.877

Table 1 summarizes the pharmacokinetic parameters obtained by fitting to a two-compartmental model. An apparent correlation can be set up between each of the coating variables and each of the various pharmacokinetic parameters studied herein. Actually, the “L”-type biphasic pharmacokinetics reflect disposition in the two compartments. In phase I, the clearance of various nanoparticles is very fast, which is in accordance with the extremely short elimination half time t1/2α. On the contrary, in phase II, the relatively long t1/2β values indicate a very slow elimination rate from the peripheral compartment (PC). The same information could be obtained from the K10 and K12 values, which represented elimination constants from the central compartment (CC) and transport constant from CC to PC, respectively. There is a positive correlation between the coating efficiency and the elimination half-lives from CC. Based on the t1/2, K10 and K12 values, the long circulation effect is highlighted by their correlation to the coating variables (PEG chain length, particle size, and coating density). Paradoxically, the K21 values, which are defined as the transport constants from PC to CC, do not follow the same rule for the coating efficiency. Although some of the most efficient long-circulating nanoparticles show faster transport from PC to CC, no general rule can be discerned. Mean residence time (MRT), calculated as AUMC/AUC, is another useful parameter to represent the overall residence of nanoparticles in the body. It is strange to find longer MRTs for non-PEGylated and less PEGylated nanoparticles, and the contribution of the later-stage resurgences of blood levels of these nanoparticles cannot be neglected.

Fig. 2 and Fig. S3 (ESI) show the live images following intravenous administration of mPEG-PCL nanoparticles. All groups show pervasive biodistribution of the nanoparticles throughout the body up to a time of 48 h. There is an obvious difference between non-PEGylated and PEGylated PCL nanoparticles. Non-PEGylated PCL nanoparticles are mostly localized to the middle abdomen region. Although there is intense localization of PEGylated nanoparticles to the same region as well, PEGylated nanoparticles tend to distribute to peripheral tissues such as the four extremities, the snout, and the genitals in enhanced concentrations. By quantification of the regions of interest (ROI) in the live images,17,18 the distribution profiles of the nanoparticles are drawn and shown in Fig. 3. All groups of nanoparticles, either PEGylated or non-PEGylated, generally follow the biphasic “L”-type distribution patterns, which are in parallel with the blood pharmacokinetic profiles. The initial stage (phase I) is characteristic of an abrupt declination in all tissues investigated herein, whereas a later steady stage (phase II) is closely followed and maintained for prolonged duration, but at much lower levels. In phase I, the overall level of nanoparticles in peripheral tissues is a few times higher than the average of the body background. Despite the overall similarity in distribution patterns, there is clear disparity between non-PEGylated and PEGylated nanoparticles. The behaviours of the least PEGylated (mPEG5k-9%-200 nm) nanoparticles is similar to that of the non-PEGylated nanoparticles, whereas those with shorter PEG chains (mPEG2k-29%-200 nm) lie between the least PEGylated (mPEG5k-9%-200 nm) and other mPEG5k nanoparticles. In phase I, the level of non-PEGylated PCL nanoparticles drops to the bottom within 0.5–1.0 h in all peripheral tissues in contrast to that of the densely PEGylated nanoparticles at about 4 h. Although there is a sudden drop for PEGylated nanoparticles as well, the rate is much slower than that obtained for non-PEGylated nanoparticles. These nanoparticles with modest to highest PEGylation are maintained at relatively high levels in peripheral tissues for as long as 48 h. The overall residence time obviously correlates positively with PEGylation density in the case of 200 nm nanoparticles. The distribution profiles of 80 nm nanoparticles appear below those of the most densely decorated (29% and 17%) nanoparticles, but above those of the least decorated (9% and mPEG2k-29%) nanoparticles. A reduction in the particle size to as small as 80 nm seems to mitigate the efficacy of modulation of the PEGylation density.

image file: c8nh00010g-f2.tif
Fig. 2 Live imaging of mPEG-PCL nanoparticles after intravenous administration to rats. To perform good comparison, the total administered dose is set to similar levels of fluorescence. Three animals are used for each group, but only images of one animal are shown here. Please refer to ESI, Fig. S3 for a whole set of images.

image file: c8nh00010g-f3.tif
Fig. 3 Fractionized semi-quantification of fluorescence of regions of interest as average radiant efficiency (ARE) (n = 3) that stands for the concentration of the nanoparticles: whole body (A), snout (B), four extremities (C), genital (D); and corresponding plots of enlargement (0–8 h) E, F, G and H, respectively.

The scanning of various organs and tissues reveals very quick real-time biodistribution of the nanoparticles in relatively higher density into the RES organs (Fig. 4). Although there is also distribution to a few other organs or tissues, the overall intensity is very low (Fig. S4, ESI). Quantification of the average radiation efficiency (ARE) reveals the tissue-based pharmacokinetics, expressed as the mean concentration of the nanoparticles in specific organs (Fig. 5 and Fig. S5, ESI). Although we cannot yet draw a statistics-based conclusion, an apparent difference is observed between groups of different coating efficiencies. In Fig. 5A, a common feature of all groups is the quick hepatic accumulation that peaks at around 1–2 h, followed by gradual elimination. It is evident that non-PEGylated and the least PEGylated groups show the highest density of nanoparticles in the liver. Since liver has the biggest volume among all the RES organs investigated herein, there is no doubt that liver is the major destination of the nanoparticles. Interestingly, the drastic accumulation of these nanoparticles is in coincidence with their rapid clearance from circulating and peripheral tissues. On the contrary, densely PEGylated nanoparticles show lower accumulation in the liver; this may be regarded as counter-evidence of the long-circulation effect. Among various PEGylated groups, there is a negative correlation between PEGylation and hepatic accumulation. The 80 nm groups show the least hepatic accumulation with total AUC a few times smaller than that of the non-PEGylated nanoparticles. The situation in the spleen is different from that in the liver. This is mainly due to pre-sequestration by the liver; there is less distribution of non-PEGylated nanoparticles to the spleen. Within the groups of the same 200 nm category, there is a positive correlation between PEGylation and splenic accumulation. This fact is in line with previous findings that PEGylation helps to evade hepatic sequestration, but may be trapped by the spleen.37–39 Seemingly, splenic accumulation is in direct correlation with long-circulating efficiency.40–42 However, it is paradoxical to find that the 80 nm groups, which have the best long-circulating effect, show the least splenic accumulation. Pulmonary accumulation is less than a degree of magnitude in comparison with hepatic or splenic accumulation. Although an obvious rule cannot be figured out, there is a tendency of incremental accumulation in the lung. The biodistribution of the nanoparticles to organs or tissues other than the RES is of even less intensity, and there is no hint of their distribution to the brain.

image file: c8nh00010g-f4.tif
Fig. 4 Ex vivo live imaging of organs dissected from SD rats after intravenous injection of P2-loaded mPEG-PCL and PCL nanoparticles. In order to curtail the total number of animals sacrificed, only one animal is used for each time point. Please refer to Fig. S4 (ESI) for a whole set of images including all time points.

image file: c8nh00010g-f5.tif
Fig. 5 Semi-quantification of fluorescence expressed as average radiant efficiency (ARE) in various RES organs: liver (A), spleen (B), lung (C). ARE stands for the average concentration of the integral nanoparticles in each organ. Please refer to Fig. S5 (ESI) for the semi-quantification data for other RES and non-RES organs.

For comparison, the pharmacokinetics of the model drug PTX was investigated following intravenous injection of PTX-loaded mPEG-PCL nanoparticles. As abovementioned, the mPEG5k-29%-80 nm and mPEG5k%-29%-200 nm groups exhibit much better long circulation properties than the PCL-200 nm group. Thus, these three formulations encapsulated with PTX were utilized to evaluate the long circulation effect based on monitoring of the plasma levels of both PTX and integral nanoparticles simultaneously. At first, PTX and P2 are successfully co-encapsulated into nanoparticles by the emulsification/solvent evaporation method, as described above. Encapsulation with PTX has a negligible effect on the particle sizes and size distribution (Table S2, ESI) as well as fluorescence stability in plasma (Fig. S6, ESI). Sustained in vitro release of PTX from all groups is observed in PBS (pH 7.4) containing 0.5% Tween 80 (Fig. 6A). A canonical protein adsorption assay confirms reduced adsorption of BSA onto nanoparticle surfaces due to the camouflaging effect of the PEG chains (Fig. 6B). After intravenous administration, superior long circulation properties of PTX-loaded PEGylated nanoparticles (PTX/mPEG5k-29%-80 nm and PTX/mPEG5k-29%-200 nm) over those of the PTX/PCL-200 nm nanoparticles are also observed (Fig. 7A and B). Similar pharmacokinetics as those of the blank nanoparticle counterparts (Fig. 1) demonstrate little influence of the loaded PTX on the behaviours of the particles per se. However, a comparison with the pharmacokinetic profiles of PTX reveals a stark disparity. Within the whole time span, there is no apparent difference between the PEGylated and non-PEGylated groups (Fig. 7D). Significantly higher plasma PTX levels of both PEGylated nanoparticles can be observed merely at 5, 15, and 30 min (nearly 2–2.5-fold) than those of PCL nanoparticles (Fig. 7E); after 1 h, there is no significant difference. Interestingly, at 5 min, only 16.4% and 17.4% of 80 nm and 200 nm PEGylated nanoparticles are removed from blood circulation (Fig. 7B), whereas the plasma PTX concentration of the corresponding formulation rapidly decreases by 82.93% (from 12.3 μg mL−1 to 2.1 μg mL−1) and 85.07% (from 13.4 μg mL−1 to 2.0 μg mL−1), respectively (Fig. 7E). This is probably ascribed to the initial burst release of PTX from the nanoparticles upon contact with blood despite the sustained release characteristics in vitro. To prove this, the release profiles of PTX in plasma were further evaluated by incubating PTX/mPEG5k-29%-200 nm and PTX/PCL-200 nm nanoparticles in plasma. The results show that over 90% of PTX is released from both nanoparticles in less than 1 h. Even after taking into account the possible release during centrifugation (0.5 h) to separate free PTX from nanoparticles, dose dumping in plasma was apparent. Similar results were obtained by other researchers and raised some confusion.14,43–46 This extraordinary example confirms that it is unreliable to explore the in vivo fate of nanoparticles by monitoring drug payloads. Although an alternative model drug may be released much slowly, it is more accurate to display the long circulation effect by directly monitoring the particles.

image file: c8nh00010g-f6.tif
Fig. 6 (A) In vitro release of PTX from PTX-loaded nanoparticles in PBS (ph 7.4) containing 0.5% tween 80; (B) BSA adsorption on the surface of PEGylated (PTX/mPEG5k-29%-200 nm) and non-PEGylated (PTX/PCL-200 nm) nanoparticles; (C) PTX release from nanoparticles in plasma. All studies were conducted in triplicate.

image file: c8nh00010g-f7.tif
Fig. 7 Pharmacokinetic profiles (n = 3) of PTX-loaded mPEG-PCL nanoparticles by monitoring intact nanoparticles (A) or PTX (D); enlargement of the pharmacokinetic profiles within 0–8 h: (B) for A, (E) for D; AUC values for pharmacokinetic profiles of integral nanoparticles (C) and PTX (F). PTX/mPEG5k-29%-200 nm vs. PTX/PCL-200 nm: *p < 0.05, **p < 0.01; PTX/mPEG5k-29%-80 nm vs. PTX/PCL-200 nm: #p < 0.05, ##p < 0.01.

Overall, the long-circulation effect is reconfirmed, but with reservations. The pharmacokinetic and biodistribution features obtained in this study deviate from the previous findings based on monitoring of drug payloads due to the strategies used to monitor integral nanoparticles. In our opinion, the findings of the effect of the crucial coating variables, such as PEG chain length and coating density, comply with previous findings.26–31 The general findings indicate that longer PEG chain lengths and heavier coating density favour longer circulation. Reduction in the particle size is found to be very efficient in prolonging the circulation time; this re-confirms previous conclusions.47–50 However, it is noteworthy that the particle size effect is more remarkable when PEGylation is less efficient, e.g. with less density. If PEGylation is satisfactorily achieved, the particle size effect may be less prominent.

Compared with the results obtained by conventional methods via analysis of the payloads, we believe that there are mainly three aspects of remarkable disparities that justify a more accurate understanding of long circulation. Contrary to PTX, various other drugs encapsulated in PEGylated nanoparticles were found to be maintained at relatively high levels for a long duration following intravenous administration.51–53 It is interesting to discover that even non-PEGylated nanoparticles circulate in blood for longer times.51,54–57 We argue that this may be due to the occulting effect of the drug-based pharmacokinetics that incorporates encapsulation and non-encapsulation as a whole. Upon removing the contribution of free drugs, a compromise in the long-circulating efficiency is inevitable. Furthermore, an obvious rebounding in the levels of blood-borne and peripheral nanoparticles is observed after the initial clearance, albeit not on a large scale, especially for non-PEGylated and less PEGylated nanoparticles. Although we still have no idea about the physiological significance of this phenomenon, its contribution to the mean residence time (MRT) of the nanoparticles in circulation should not be ignored (Table 1). This phenomenon evokes our interest to investigate the underlying mechanisms and explore the physiological significance to support potential applications. In addition, it has long been established that long circulation of NPs is directly related to their enhanced accumulation in various RES and non-RES organs and tissues as a direct result of perfusion and sequestration. For the case of splenic filtration, we found that the most efficient long-circulating particles (80 nm categories) achieved the least splenic targeting. Herein, the particle size seems to outweigh the efficiency of PEGylation.

The physiological significance of long circulation is not limited to prolonged residence time of the particles in circulation. Long circulation provides more opportunities for nanoparticles to distribute to non-RES tissues, circumventing the phagocytic uptake barriers that dominate the RES organs such as liver, spleen, lung, and kidney. By live imaging, the PEGylated nanoparticles are found to be distributed to peripheral tissues in accentuated levels, normally 3–5 times of the bulk background, which is in line with our previous results of mPEG-PLA polymeric micelles simulating a commercial product—Genexol.11 Once again, these findings suggest that there may be some link between the enhanced peripheral accumulation and the so-called hand-foot syndrome (HFS). Several clinical studies with PEGylated liposomal doxorubicin have reported severe HFS with a total occurrence of 50% and 20% of severe grade three HFS.58–60 This newly found toxicity might be ascribed to the long circulation effect as a result of PEGylation. Although the details differ, the overall nanoparticle level vs. time profiles follow a similar pattern as blood pharmacokinetics. This observation reveals important information to correlate the blood pharmacokinetics with enhanced peripheral accumulation as a direct consequence of blood perfusion.

We conclude that the previous perception of the long-circulation effect by PEGylation may be biased due to indiscriminative quantification of the drug payloads. Reassessment by monitoring of integral nanoparticles justifies a more accurate understanding of the concept of long circulation as well as the behaviours of the particles.

This study was financially supported by the National Natural Science Foundation of China (81573363, 81690263, 21372063), the Science and Technology Commission of Shanghai Municipality (14JC1490300), and the National Key Basic Research Program (2015CB931800).

Conflicts of interest

There are no conflicts to declare.

Notes and references

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Electronic supplementary information (ESI) available: Detailed experimental procedures; more characterization data. See DOI: 10.1039/c8nh00010g
These authors contributed to this article equally.

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