Apolipoprotein E Peptide-Directed Chimeric Polymersomes Mediate

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Apolipoprotein E Peptide-Directed Chimeric Polymersomes Mediate an Ultrahigh-Efficiency Targeted Protein Therapy for Glioblastoma Yu Jiang, Jian Zhang, Fenghua Meng, and Zhiyuan Zhong ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b05265 • Publication Date (Web): 05 Nov 2018 Downloaded from http://pubs.acs.org on November 5, 2018

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Apolipoprotein E Peptide-Directed Chimeric Polymersomes Mediate an Ultrahigh-Efficiency Targeted Protein Therapy for Glioblastoma Yu Jiang, Jian Zhang*, Fenghua Meng, Zhiyuan Zhong*

Biomedical Polymers Laboratory, and Jiangsu Key Laboratory of Advanced Functional Polymer Design and Application, College of Chemistry, Chemical Engineering and Materials Science, and State Key Laboratory of Radiation Medicine and Protection, Soochow University, Suzhou, 215123, P. R. China. E-mail: [email protected] (J. Zhang), [email protected] (Z. Zhong).

ABSTRACT Inability to cross blood brain barrier (BBB) refrains nearly all chemotherapeutics and biotherapeutics from effective treatment of brain tumors, rendering little improvement of patient survival rates to date. Here, we report that apolipoprotein E peptide (ApoE, (LRKLRKRLL)2C), which specifically binds to low-density lipoprotein receptor members (LDLRs), mediates superb BBB crossing and highly efficient glioblastoma (GBM)-targeted protein therapy in vivo. The in vitro BBB model studies reveal that ApoE induces 2.2-fold better penetration of the immortalized mouse brain endothelial cell line (bEnd.3) monolayer for chimeric polymersomes (CP) compared with Angiopep-2, a best-known BBB crossing peptide used in clinical trials for GBM therapy. ApoE-installed CP (ApoE-CP) carrying saporin (SAP) displays highly specific and potent antitumor effect toward U-87 MG cells 1

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with a low IC50 of 14.2 nM SAP. Notably, ApoE-CP shows efficient BBB crossing as well as accumulation and penetration in orthotopic U-87 MG glioblastoma. The systemic administration of SAP-loaded ApoE-CP causes complete growth inhibition of orthotopic U-87 MG GBM without eliciting any observable adverse effects, affording markedly improved survival benefits. ApoE peptide provides an ultrahigh-efficiency targeting strategy for GBM therapy.

KEYWORDS: brain tumor, blood brain barrier, protein delivery, nanomedicines, targeted therapy

Glioblastoma (GBM) is the most invasive intracranial primary tumor that remains incurable to date.1-3 Inability to cross blood brain barrier (BBB) refrains nearly all chemotherapeutics and biotherapeutics from effective treatment of GBM patients.4 It is found that a plethora of receptors, such as low-density lipoprotein (LDL) receptor family (LDLRs), transferrin receptor, and insulin receptor are highly expressed on BBB,5 which enable receptor-mediated transcytosis (RMT) of cargos through BBB.6-8 In particular, three members of LDLRs, i.e. LDL receptor (LDLR) and LDLR-related protein 1 and 2 (LRP1 and LRP2), demonstrate significant up-regulation along with the development of glioblastoma.9 Notably, these three receptors are also overexpressed on GBM cells,10, 11 which renders LDLRs an ideal target for GBM therapy. In the past years，peptides targeting LDLRs, including Angiopep-2 (ANG),12 ApoB (3371-3409),13 and Peptide-22,14, 15 which target LRP1, LRP2 and LDLR, respectively, have been explored for delivering anticancer drugs to GBM. ANG, as a most advanced GBM targeting peptide, is currently under clinical development.16-18 The functionalization of nanoparticles with ANG showed clearly enhanced BBB transcytosis.19, 20 Notably, apolipoprotein E3 has shown efficient GBM-targeting delivery of siRNA.21 Several 2


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apolipoprotein E derived peptides have recently been developed for crossing BBB,22-25 among which a tandem dimer sequence of the receptor-binding domain of apolipoprotein E, ApoE peptide, was reported to significantly improve brain delivery of therapeutics without interfering with endogenous apolipoprotein E.26 The high brain delivery efficiency of ApoE peptide may be ascribed to its high affinity to multiple LDLRs, including LDLR, LRP1 and LRP2.27 Here, we report that ApoE directed superb BBB crossing and highly efficient GBM-targeted protein therapy in vivo (Scheme 1). Protein therapeutics has recently emerged as a next-generation treatment modality for cancers.28, 29 We found that chimeric polymersomes (CP) are an ideal carrier system for protein delivery,30-32 and ANG-functionalized CP boosts protein therapy for GBM.33 Interestingly, our results show that ApoE peptide-decorated CP (ApoE-CP) further enhances BBB transcytosis and GBM-targeting ability over ANG counterparts, leading to effective inhibition of intracranial U-87 MG human GBM xenografts in nude mice and significantly increased survival time. ApoE peptide emerges as a highly interesting ligand for targeted GBM therapy.

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Scheme 1. Schematic presentation of ApoE peptide-decorated chimeric polymersomes (ApoE-CP) for targeted protein therapy for GBM.a

a

(i) ApoE peptide effectively crosses BBB via multi-receptor (including LDLR, LRP1 and

LRP2) mediated transcytosis; (ii) ApoE peptide mediates specific and efficient uptake of ApoE-CP in GBM cells; and (iii) ApoE-CP quickly releases protein payloads into the cytoplasm of GBM cells, triggered by their high glutathione (GSH) concentration, resulting in potent and selective inhibitory effect to GBM cells.

RESULTS AND DISCUSSION Preparation and characterization of ApoE-CP-SAP ApoE-CP-SAP was readily prepared from poly(ethylene glycol)-b-poly(dithiolane trimethylene carbonate-co-trimethylene carbonate)-b-polyethylenimine triblock copolymer 4


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(PEG-P(DTC-TMC)-PEI, Mn = 5.0-(2.2-14.6)-1.8 kg/mol) and ApoE peptide-modified PEG-P(DTC-TMC) diblock copolymer (ApoE-PEG-P(DTC-TMC), ApoE functionality = 93%, Mn = 7.5-(1.9-15.2) kg/mol). As reported previously,34, 35 to potentiate targeting effect of ApoE ligand, ApoE-PEG-P(DTC-TMC) was devised with a longer PEG than its counterpart in PEG-P(DTC-TMC)-PEI (7.5 versus 5.0 kg/mol). For SAP loading, by increasing theoretical protein loading contents (PLC) from 5 to 15 wt.% (Table S1), the PLC of ApoE-CP for SAP increased from 4.6 to 11.5 wt.% accordingly due to the strong interactions between SAP and the PEI moieties of polymersomes.36, 37 In addition, the size of ApoE-CP-SAP varied from 80 to 86 nm according to the increase of PLC (Table S1) Furthermore, ApoE-CP-SAP possesses narrow size distribution (Figure 1A) and a vesicular morphology (Figure 1B). We found that ApoE-CP-SAP exhibits nearly identical distribution to empty ApoE-CP (Figure S3), indicating that loading of SAP has little influence on polymersomes. ApoE-CP-SAP was robust against dilution (with final concentration of 0.01 mg/mL) and 10% fetal bovine serum (FBS) (Figure S4A). However, micron-scale particles were observed under reductive condition (10 mM GSH, pH 7.4 and 37 oC) for 12 h (Figure S4B). ApoE-CP-SAP displayed essentially neutral zeta potentials. The non-targeted control, CP-SAP, prepared solely from PEG-P(DTC-TMC)-PEI, demonstrated similar PLC, size distribution and zeta potentials to ApoE-CP-SAP (Table S1). The release of SAP from ApoE-CP-SAP was below 15% in 24 h under non-reductive condition (Figure S4C), supporting that ApoE-CP-SAP is robust. In contrast, over 85% SAP was released under a reductive condition, in line with fast reduction-triggered protein release from ApoE-CP-SAP.

Receptor-mediated GBM cell uptake and BBB transcytosis To investigate whether LDLRs are overexpressed in the BBB endothelium and GBM cells, we have performed Western blot assays in bEnd.3 cells and U-87 MG cells. The results

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corroborated that bEnd.3 cells overexpress LRP1 and LRP2 while U-87 MG GBM cells overexpress LRP1, LRP2, and LDLR (Figure 1C). In comparison, none of LDLRs is overexpressed on normal HA1800 astrocytes. Hence, ApoE peptide, which is able to target to LDLRs, is a potentially interesting ligand to achieve high-efficiency targeting effect toward GBM. LDLRs-mediated U-87 MG cellular uptake was studied by flow cytometry using Cy5-labled ApoE-CP at varying ApoE surface densities from 10 to 30 mol.%. Figure 1D shows that ApoE-CP with 20 mol.% ApoE (denoted as ApoE20-CP) had ca. 4-fold enhanced uptake compared with CP (non-targeted control) in U-87 MG cells. Notably, ApoE20-CP had ca. 2-fold better uptake by U-87 MG cells than ANG-modified CP.33 Increasing ApoE density to 30% didn’t further improve cellular uptake. Pre-treating U-87 MG cells with free ApoE resulted in markedly decreased uptake of ApoE20-CP. These results verify that ApoE-CP is mainly internalized by U-87 MG cells via receptor-mediated endocytosis. In the following, ApoE20-CP was selected for further investigation. Unless otherwise stated, ApoE-CP refers to ApoE20-CP. The in vitro BBB transcytosis capability of ApoE-CP was evaluated by a transwell study (Figure 1E). ApoE-CP showed 4.8-fold better transport ratio than non-targeted CP (p < 0.001) and 2.2-fold better than ANG-modified chimeric polymersomes (ANG-CP) (p < 0.001) (Figure 1F). This indicates, probably, the best BBB transport ability among all the reported BBB targeting peptides.9, 19, 38, 39 As expected, the in vitro BBB transport ratio of ApoE-CP was greatly decreased by pretreating bEnd.3 monolayer with free ApoE peptide (100 μg/mL), indicating that the BBB transcytosis of ApoE-CP is mainly mediated by LDLRs.

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Figure 1. (A) The typical size distributions of SAP-loaded polymersomes, ApoE-CP-SAP and CP-SAP. (B) TEM image of ApoE-CP-SAP. (C) Expression levels of LDLRs (including LRP1, LDLR, and LRP2) on cell lines of U-87 MG-Luc, bEnd.3 and normal HA1800 astrocyte determined by Western blot. (D) Influence of ApoE surface densities on endocytosis of ApoE-CP by U-87 MG cells measured by flow cytometry. (E) Illustration of Cy5-labeled polymersomes crossing BBB model established using bEnd.3. (F) The in vitro BBB model transport ratios (%) of Cy5-labeled CP, ANG-CP, and ApoE-CP following 24 h incubation. 7


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Blockade experiments in C and D were conducted by pre-treating U-87 MG cells with free ApoE (100 μg/mL, 30 minutes) before ApoE-CP incubation.

In vitro antitumor activity and intracellular protein release of ApoE-CP-SAP The in vitro anti-GBM potency of ApoE-CP-SAP toward U-87 MG-Luc GBM cells were evaluated by MTT assays. Figure 2A indicates that blank ApoE-CP and CP, up to 200 μg/mL, were nontoxic to GBM cells. In contrast, ApoE-CP-SAP showed potent antitumor effect toward U-87 MG-Luc cells with a half-maximum inhibitory concentration (IC50) of 14.2 nM SAP (Figure 2B), comparable to that of immunotoxins.40, 41 The non-targeted CP-SAP showed significantly lower antitumor potency, in which more than 75% GBM cells were viable at 40 nM SAP. As anticipated, free SAP didn’t show obvious antitumor activity even at 40 nM, owing to its lack of cell inserting subunit.41 It was reported that SAP would induce up-regulation of specific apoptosis-related proteins, including pERk1/2 and Bax.42 Notably, obvious up-regulation of both pERk1/2 and Bax was detected in ApoE-CP-SAP incubated U-87 MG-Luc cells (Figure 2C). Interestingly, CP-SAP treated cells also showed slightly higher Bax expression. In contrast, free SAP, blank ApoE-CP and CP treated U-87 MG-Luc cells all showed a low expression of pERk1/2 and Bax, similar to those with PBS. Next, FITC-labeled CC (CC(FITC)), a model protein with similar molecular weight as well as isoelectric point to that of SAP, was encapsulated into ApoE-CP to investigate the LDLRs-mediated endocytosis of ApoE-CP by U-87 MG cells. Figure 2D reveals that ApoE-CP-CC(FITC) treated U-87 MG-Luc cells exhibited strong cytoplasmic FITC fluorescence, supporting fast cellular uptake and intracellular release of proteins. In contrast, non-targeted CP-CC(FITC) treated cells had obviously less FITC fluorescence and no detectable FITC fluorescence came out from free protein incubated cells. It is clear that

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ApoE-CP-SAP could efficiently deliver payloads to cytoplasm of U-87 MG-Luc cells, resulting in selective and potent anti-GBM effects.

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Figure 2. (A) MTT assays of blank ApoE-CP and CP in U-87 MG-Luc cells. (B) In vitro anti-GBM efficacy of ApoE-CP-SAP and CP-SAP to U-87 MG-Luc cells. The data were given as mean ±SD (n = 4). (C) Western blot analyses of pERk1/2 and Bax expressions in U-87 MG-Luc GBM cells treated with different formulations for 4 h. GAPDH was used as a control. (D) CLSM images of U-87 MG cells treated with ApoE-CP-CC(FITC), CP-CC(FITC) or free CC(FITC) (CC(FITC) dosage: 1.0 μg/mL). The CLSM images were presented in the order of, from left to right, cell nuclei stained by DAPI (blue), cytoskeleton stained by phalloidin-rhodamine B (red), CC(FITC) fluorescence (green), and overlays of the three images. Scale bars: 10 μm.

The penetrating and tumor targeting efficacy of ApoE-CP evaluated through BBTB/U-87 MG tumor spheroids co-culture model. To evaluate the penetrating and GBM targeting ability of ApoE-CP, we have established BBTB/U-87 MG tumor spheroids co-culture model as reported.43 ApoE-CP was labeled with Cy5. Interestingly, strong Cy5 fluorescence was detected inside the tumor spheroids treated with Cy5-labeled ApoE-CP (Figure 3A), supporting effective GBM penetration of ApoE-CP. In contrast, tumor spheroid treated with non-targeting Cy5-CP displayed faint Cy5 fluorescence and only in the superficial area (Figure 3B). These results verify that the functionalization of CP with ApoE peptide can significantly improve its BBTB penetration and GBM targeting ability.

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Figure 3. The penetrating and tumor targeting efficacy of Cy5-labeled ApoE-CP (A) and Cy5-labeled CP (B) evaluated through BBTB/U-87 MG tumor spheroids co-culture model. The images were taken using multi-level scan of confocal microscope. Scale bars: 200 m.

Biodistribution and in vivo pharmacokinetics It has been recognized that for in vivo targeting, ligand density is of critical importance.44 To optimize the surface density of ApoE peptide, we investigated the accumulation of DiR-loaded ApoE-CP in the orthotopic human U-87 MG-Luc tumor mouse model. Figure 4A shows that intracranial U-87 MG-Luc GBM was successfully established. Interestingly, whole body near infrared imaging revealed that in sharp contrast to CP, ApoE-CP with 10, 20 and 30 mol.% ApoE all could deliver DiR to GBM tumor (Figure 4A). The ex vivo fluorescence 11


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images displayed clearly that ApoE20-CP achieved the best GBM accumulation (Figure 4B). Semi-quantitative fluorescence analysis (Figure 4C) further confirmed that the GBM accumulation of ApoE20-CP was significantly higher than that of ApoE30-CP, ApoE10-CP or CP. The pharmacokinetics studies using 125I-labeled SAP showed that SAP-ApoE-CP and SAP-CP had similarly long elimination half-life (t1/2β = ~ 5 h) (Figure 4D), indicating that ApoE modification has little influence on the pharmacokinetics of CP. In contrast, free SAP was rapidly cleared from the systemic circulation with a short t1/2β of 1.1 h. The anti-GBM efficacy of a drug is largely limited by its poor cerebral microvascular permeability and GBM tissue penetration.45, 46 Here, we investigated the BBB permeability and tumor penetration of ApoE-CP-CC(Cy5). Interestingly, strong Cy5 fluorescence was observed from both boundary and center of the tumor sections taken from ApoE-CP-CC(Cy5) treated animals (Figure 4E). CP-CC(Cy5) treated group showed some Cy5 fluorescence at the boundary of tumor and brain parenchyma, likely due to partly compromised BBB along with the development of GBM. This “passive” accumulation of CP-CC(Cy5) gave no Cy5 fluorescence in the GBM center. As anticipated, no Cy5 fluorescence was detected throughout the whole tumor sections from free Cy5-CC treated mice. The above results confirm that ApoE-CP can cross BBB and penetrate deep into GBM lesion in vivo.

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Figure 4. (A) The bioluminescence images of intracranial U-87 MG-Luc GBM mouse model. and real-time whole-body DiR fluorescence imaging of GBM-bearing animals at 24 hours after intravenous injection of CP-DiR and ApoE-CP-DiR with varying ApoE surface densities of 10, 20 and 30 mol.%. (B) Ex vivo DiR fluorescence images of excised organs, including 13


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GBM-bearing brains, from mice sacrificed at 24 h post-injection. (C) Semi-quantitative analysis of DiR fluorescence intensity in GBM sections. (D) In vivo pharmacokinetics of 125

I-labeled SAP, 125I-labeled SAP-loaded ApoE-CP and 125I-labeled SAP-loaded CP-SAP in

mice. (E) Fluorescence images of GBM sections taken from animals treated with free CC(Cy5), CP-CC(Cy5) or ApoE-CP-CC(Cy5) at 24 hours post-injection. The lower part (under the yellow dash line and indicated by an arrow) in each section image indicates the GBM area. Scale bar: 50 μm.

Targeted protein therapy for intracranial GBM The anti-GBM potency of ApoE-CP-SAP was investigated in intracranial U-87 MG GBM-bearing mouse model. Interestingly, bioluminescence monitoring clearly demonstrated the growth inhibition of ApoE-CP-SAP (SAP dosage: 8.33 nmol SAP equiv./kg) against intracranial GBM over a period of 20 days (Figure 5A). Doubling ApoE-CP-SAP dosage to 16.66 nmol SAP equiv./kg led to complete inhibition of GBM progression. In comparison, non-targeted CP-SAP caused apparently less effective GBM inhibition than ApoE-CP-SAP. It should further be noted that free SAP at 16.66 nmol SAP equiv./kg had no treatment effect to U-87 MG-Luc GBM. The semi-quantitative bioluminescence analyses confirmed that U-87 MG-Luc GBM was highly aggressive and ApoE-CP-SAP potently inhibited GBM growth at 8.33 and 16.66 nmol SAP equiv./kg (Figure 5B). Figure 5C shows that ApoE-CP-SAP had a tumor inhibition rate (TIR) of 91.2 and 94.5 at a SAP dosage of 8.33 and 16.66 nmol SAP equiv./kg, respectively. Along with the progression of orthotopic GBM, animals’ health conditions deteriorate quickly. As a result, the mice treated with PBS or free SAP displayed fast and significant body weight loss (~ 30%) in 22 days, due to aggressive invasion of GBM into the brain (Figure 5D). The similar body weight loss profile observed for free SAP and PBS indicates that free SAP has no therapeutic effect and also does not induce systemic

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toxicity. Strikingly, animals of both ApoE-CP-SAP treated groups didn’t show any body weight decrease, confirming effective suppression of GBM invasion. This is in sharp contrast to reported chemical drug-based nanomedicines that are typically associated with significant systemic toxicity.47-49 Intriguingly, Kaplan–Meier survival analysis revealed that ApoE-CP-SAP remarkably extended the survival time of GBM-bearing animals, in which median survival time of 52 and 59 days was achieved at 8.33 and 16.66 nmol SAP equiv./kg, respectively (Figure 5E). In contrast, animals in free SAP and PBS groups all died in 24 days. The non-targeted CP-SAP groups had only slight improvement on the mice survival rate. Notably, ApoE-CP-SAP brings about far better survival benefits than chemotherapeutics-based nanomedicines recently reported for orthotopic GBM bearing mice (refs),50-52 in which comparably short median survival times of 28 to 37 days were observed for doxorubicin and paclitaxel-based nano-formulations. The superior treatment efficacy of ApoE-CP-SAP compared with targeted chemotherapy is likely associated with its ApoE-mediated efficient BBB penetration and selective GBM cell uptake, as well as high potency and low adverse effects of protein drug over chemotherapeutics. We have made a comparison between ApoE and ANG for BBB crossing and GBM targeting (Table 1). ANG-CP had the same optimal peptide density (20 mol.%) and similar protein loading content to ApoE-CP (8.8 wt.% vs. 8.5 wt.%).33 Interestingly, ApoE-CP exhibits over 2 times better BBB transcytosis and GBM accumulation than ANG-CP. Moreover, ApoE-CP-SAP demonstrates also a lower IC50 compared with ANG-CP-SAP. In accordance, ApoE-CP-SAP led to clearly better survival benefit than ANG-CP-SAP.33 This multi-receptor targeting ability of ApoE peptide appears to be a better strategy than single-receptor targeting modality (e.g. ANG) for GBM treatment.

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Figure 5. In vivo anti-GBM efficacy of ApoE-CP-SAP (dosage: 8.33 or 16.66 nmol SAP equiv./kg) in orthotopic human U-87 MG-Luc GBM-bearing mouse model (n = 7). (A) Orthotopic GBM bioluminescence images on day 10, 15 and 20. (B) Semi-quantitative analysis of mean bioluminescence intensity of U-87 MG-Luc GBM. (C) Mean tumor inhibition rate. (D) Body weight changes of mice in 26 days. (E) Kaplan–Meier survival analysis of mice (median survival time: 59, 52, 34, 32, 23, 21 days for I, II, III, IV, V and VI, respectively). Statistical analysis: ApoE-CP-SAP (16.66 nmol SAP equiv./kg) vs. ApoE-CP-SAP (8.33 nmol SAP equiv./kg), p < 0.01; ApoE-CP-SAP (16.66 nmol SAP equiv./kg) vs. CP-SAP (16.66 nmol SAP equiv./kg) and free SAP (16.66 nmol equiv./kg), p < 0.001; ApoE-CP-SAP (8.33 nmol SAP equiv./kg) vs. CP-SAP (16.66 nmol SAP equiv./kg), CP-SAP (8.33 nmol SAP equiv./kg), and free SAP (16.66 nmol equiv./kg), p < 0.001 (Kaplan-Meier analysis, log-rank test). (F) TUNEL assays of the GBM tissues, with apoptotic cells shown in green and DAPI-stained cell nuclei shown in blue, following different treatments. Scale bar: 50 μm. Table 1. Comparison of ApoE and Angiopep-2 peptides in BBB crossing and GBM targeting. CP

Transport ratio of Cy5-labeled CPa (%)

IC50 of SAP-loaded CPb (nM)

Tumor accumulation of DiR-loaded CP (% ID/g)

Survival time of mice treated with SAP-loaded CP (day)

ApoE-CP

27.6

14.2

4.26

52

ANG-CP

12.8

30.2

1.71

43

a)

Determined by in vitro BBB model established by bEnd.3 cell line;

b)

Determined by MTT assays;

c)

Determined by fluorometry;

The apoptosis induction of ApoE-CP-SAP was further verified by TUNEL assays. Figure 5F shows clearly that tumor sections taken from ApoE-CP-SAP treated animals demonstrated 17


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obviously more apoptotic GBM cells (shown in green) than those with CP-SAP, free SAP and PBS. It has to be pointed out that the mild anti-GBM effect of non-targeted CP-SAP is mainly attributed to its passive accumulation in GBM boundary adjacent as a result of partly compromised BBB along with GBM development. The histological analyses using H&E staining indicated that both ApoE-CP-SAP and CP-SAP did not induce damage to the major organs (Figure S5). We further performed hematology and blood biochemistry analyses of mice treated with ApoE-CP-SAP. The results showed clearly that ApoE-CP-SAP treatment had little inﬂuence on the hematological parameters (Figure S6A) as well as liver and kidney functions (Figure S6B). It is clear that ApoE-directed and reduction-sensitive chimeric polymersomes mediate an ultrahigh-efficiency, nontoxic, and targeted protein therapy for GBM.

CONCLUSION We demonstrate that apolipoprotein E peptide-directed and reduction-sensitive chimeric polymersomes (ApoE-CP) mediate an ultrahigh-efficiency targeted protein therapy for orthotopic human GBM xenografts in nude mice. ApoE-CP has many advantages: (i) they can effectively cross BBB via multi-receptor (including LDLR, LRP1 and LRP2) mediated BBB transcytosis; (ii) they show specific and highly efficient uptake in GBM cells via multi-receptor (including LDLR, LRP1 and LRP2) mediated endocytosis; (iii) they exhibit high loading of protein therapeutics; and (iv) they rapidly release proteins into the cytoplasm of GBM cells, leading to high-efficacy inhibition of GBM growth. This multi-receptor targeting of ApoE peptide appears to be a better strategy than single-receptor targeting modality. ApoE-targeted and reduction-sensitive chimeric polymersomes have great potential for safe and potent intracranial GBM therapy by therapeutic proteins.

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METHODS Receptor expression evaluation The expression levels of LDLRs (including LDLR, LRP1 and LRP2) on U-87 MG-Luc GBM, bEnd.3 and normal astrocyte cells (HA1800) were measured by Western blot assays. In brief, approximately 3  106 cells were homogenized with 0.15 mL of radio-immunoprecipitation assay buffer (RIPA) containing protease inhibitor for 20 minutes at 4 °C. After homogenization, samples were centrifuged (12,000 rpm, 15 minutes, 4 °C) and the supernatants were quantified for protein concentrations by bicinchoninic acid (BCA) protein assay kit and then, equal amounts of total protein samples of different cells were mixed with sample loading buffer (5 ) and added to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The gels were transferred to polyvinylidene difluoride membranes, followed by membrane blockade with 5% bovine serum albumin (BSA) at room temperature and incubation at 4 °C with the primary antibody rabbit anti-human LRP1, LDLR and LRP2 (1:1000 dilution) overnight and cultured with secondary antibody phycoerythrin labeled goat anti-rabbit IgG (1:10,000 dilution). After washing with Tris-buffered saline with Tween-20 (TBST) for three times, the fluorescence signals were taken using Super Signal ECL on a Bio-Rad ChemiDoc MP System. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cultured with GAPDH antibody (1:1000 dilution), was used as a control in this analysis. Confocal study CC(FITC) was encapsulated into polymersomes to assess the capability of endocytosis and cytoplasmic protein release of ApoE-CP. U-87 MG-Luc GBM cells were cultured on glass cover slides embedded in 24-well plates (1 × 105 cells/well) for 24 hours. CC(FITC) loaded polymersomes, including ApoE-CP-CC(FITC), CP-CC(FITC) and CC(FITC) (CC concentration: 40 μg/mL), were added to each well and cultured for 4 hours. After incubation, 19


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the sample-containing medium was replaced by equal volume of fresh medium, followed by another 4 hours incubation. After removing the medium, the cells were fixed by 4% paraformaldehyde, the cytoskeleton was stained by Rhodamine B labeled phalloidin, and the cell nuclei were stained with 4′,6-diamidino-2- phenylindole (DAPI). The fluorescence images were taken by a confocal laser scanning microscope (CLSM, Leica, TCS SP5). Western blot assay U-87 MG-Luc cells were seeded in 6-well plates (2 × 105 cells/well) and cultured for 24 hours. The cells were incubated with SAP, CP-SAP, ApoE-CP-SAP or equivalent vector for 4 hours. After that, the medium was replaced with fresh medium and the cells were cultured for another 44 hours. The cells were lysed with 0.1 mL RIPA buffer supplemented with protease and phosphatase inhibitors. The protein concentration of different samples was measured by BCA kit. Equal amounts of total proteins from samples were mixed with loading buffer and boiled for 5 minutes and detached on a 10% SDS-PAGE, then transferred to a polyvinylidene difluoride membrane. The membranes were blocked with 5% bovine serum albumin (BSA) for 1 hours at room temperature. After incubation overnight at 4 °C with the corresponding primary antibody rabbit anti human pERk1/2, Bax（1:1000 dilution, the membranes were treated with secondary antibody goat anti-rabbit IgG (1:10000 dilution) at 25 oC for 1 hours. After washing with TBST for three times, the fluorescence signals were monitored using Super Signal ECL (Pierce, U.S.A.). Real-time GBM targeting of ApoE-CP Orthotopic GBM bearing mice were used to evaluate the in vivo targeting ability of polymersomes modified with different ApoE surface densities. 13 days post-implantation, Animals with similar tumor burden were randomly divided into four groups, and treated with 1,1’-dioctadecyltetramethyl indotricarbocyanine iodide (DiR)-loaded CP, ApoE10-CP, ApoE20-CP and ApoE30-CP (4 µg DiR equiv./animal), respectively. At predetermined time 20


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points (2, 6, 12 and 24 hours) post-injection, animals were anesthetized with pentobarbital sodium solution and whole body near-infrared fluorescence images were acquired (IVIS Lumina II, λEx/λEm = 747 nm/774 nm). At 24 hours time point, GBM bearing brains and other main organs (including heart, liver, spleen, lung and kidney) were collected for ex vivo fluorescence imaging and quantitative analysis. Biodistribution of ApoE-CP in orthotopic GBM To study the tumor homing of protein-loaded polymersomes, sulfo-cyanine5 amine-labeled cytochrome C (CC(Cy5)) loaded polymersomes (0.1 mg Cy5 equiv./kg) were intravenously injected into the nude mice bearing orthotopic human GBM xenografts (13 days post-implantation). After 8 hours, the mice were anesthetized with pentobarbital sodium and sacrificed to collect GBM bearing brains. The excised brains were fixed in paraformaldehyde (4%, 48 hours), dehydrated with 10% and then 30% sucrose solution, and further embedded in paraffin (Sakura, Torrance, CA, USA) to prepare brain slices. After staining of cell nucleus with DAPI (1 g/mL) for 10 minutes, a clear border between GBM and normal brain tissue was observed owing to the visual difference of the cell nuclei of GBM (pyknotic) and normal brain tissue (spares). Finally, GBM slices were visualized with a CLSM. Anti-GBM efficacy 10 days after implantation, orthotopic GBM bearing mice were randomly divided into six groups according to bodyweight and tumor bioluminescence and i.v. injected with PBS, SAP (16.66 nmol/kg), CP-SAP (8.33 nmol SAP equiv./kg), CP-SAP (16.66 nmol SAP equiv./kg), ApoE-CP-SAP (8.33 nmol SAP equiv./kg) or ApoE-CP-SAP (16.66 nmol SAP equiv./kg) on day 10, 13, 16 and 19 after implantation. To monitor the in vivo tumor inhibition of different formulations, the bioluminescence of animals was monitored (IVIS Lumina II) on day 10, 15, 20 after implantation via i.v. injection of luciferin potassium salt (75 mg/kg). The relative bioluminescence intensity of GBM was defined as I/I0 (I0 is the mean bioluminescence 21


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intensity of GBMs at day 10). Mice were weighed and normalized to their initial weights on day 10 post-implantation. On day 25 post-implantation, one representative mouse from each group was sacrificed and major organs, including GBM bearing brains, were excised for histological analysis. For PBS and free SAP groups, one mouse was sacrificed on day 20, due to a short survival time. The excised tissues were fixed by paraformaldehyde and embedded in paraffin. In addition, the brain tissue was frozen sectioned for terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) and observed using a CLSM. The major organs such as heart, liver, spleen, lungs and kidney were stained using hematoxylin and eosin. The histological sections were observed under an optical microscope. The other 7 animals of each group were further monitored and recorded for survival analysis. Statistical analysis Data are expressed as mean ±standard deviation. Significance among groups is analyzed by one-way analysis of variance (ANOVA) with Bonferroni correction being used for comparison between individual groups. Survival data significance is calculated by Log-rank test. *p

Apolipoprotein E Peptide-Directed Chimeric Polymersomes Mediate

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