Blood circulation-prolonging peptides for engineered nanoparticles

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Blood circulation-prolonging peptides for engineered nanoparticles identified via phage display peipei jin, Rui Sha, Yunjiao Zhang, Liu Liu, Yunpeng Bian, Jing Qian, Jieying Qian, Jun Lin, Nestor Ishimwe, Yi Hu, Wenbin Zhang, Yanchun Liu, Shiheng Yin, Li Ren, and Longping Wen Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.8b04007 • Publication Date (Web): 07 Feb 2019 Downloaded from http://pubs.acs.org on February 7, 2019

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ACS Paragon Plus Environment

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Blood circulation-prolonging peptides for engineered nanoparticles identified via phage display Peipei Jin1,2,7#, Rui Sha6#, Yunjiao Zhang1,2,4#, Liu Liu6, Yunpeng Bian6, Jing Qian2,3, Jieying Qian1,3, Jun Lin6, Nestor Ishimwe6, Yi Hu 6, Wenbin Zhang1,4, Yanchun Liu7, Shiheng Yin8, Li Ren5* & Long-ping Wen1,2,3* 1Guangzhou

First People’s Hospital, School of Medicine & Institutes for Life Sciences, South China University of Technology,

Guangzhou 510006, China 2National

Engineering Research Center for Tissue Restoration and Reconstruction, South China University of Technology,

Guangzhou 510006, China 3Key

Laboratory of Biomedical Engineering of Guangdong Province, and Innovation Center for Tissue Restoration and

Reconstruction, South China University of Technology, Guangzhou 510006, China 4Key

Laboratory of Biomedical Materials and Engineering of the Ministry of Education, South China University of Technology,

Guangzhou 510006, China 5School

of Materials Science and Engineering, South China University of Technology, Guangzhou, China

6School

of Life Sciences, University of Science and Technology of China, Hefei, Anhui, China

7The

Key Laboratory of Energy-Efficient Functional Ceramics and Applied Technology of Guangdong Province, Guangzhou

Redsun Gas Applications Co., LTD, Guangzhou, China 8Analytical

#:

and Testing Center, South China University of Technology, Guangzhou, 510640, PR China

These authors contributed equally to this work

Correspondence and requests for materials should be addressed to L.P.W. (email: [email protected], phone number: 020-39380985) or L.R. (email: [email protected], phone number: 020-39380255)

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Abstract

Sustaining blood retention for theranostic nanoparticles is a big challenge. Various approaches have been attempted and have demonstrated some success, but limitations remain. We hypothesized that peptides capable of increasing blood residence time for M13 bacteriophage, a rod-shaped nanoparticle self-assembled from proteins and nucleic acids, should also prolong blood circulation for engineered nanoparticles. Here we demonstrate the feasibility of this approach by identifying a series of blood circulation-prolonging (BCP) peptides through in vivo screening of an M13 peptide phage display library. Intriguingly, the majority of the identified BCP peptides contained an arginine-glycine-aspartic acid (RGD) motif, which was necessary but insufficient for the circulation-prolonging activity. We further demonstrated that the RGD-mediated specific binding to platelets was primarily responsible for the enhanced blood retention of BCP1. The utility of the BCP1 peptide was demonstrated by fusion of the peptide to human heavy-chain ferritin (HFn), leading to significantly improved pharmacokinetic profile, enhanced tumor cell uptake and optimum anti-cancer efficacy for doxorubicin encapsulated in the HFn nanocage. Our results provided a proof-of-concept for an innovative yet simple strategy, which utilizes phage display to discover novel peptides with the capability of substantially prolonging blood circulation for engineered theranostic nanoparticles.

Key Words Nanoparticles; Blood circulation-prolonging; Peptide; Phage display; Heavy-chain ferritin 2


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Engineered nanoparticles, intended for diagnostic or therapeutic intervention of human diseases, hold great promise in medicine, but their in vivo applications also face enormous challenges. One of the critical issues is the short blood residence time, as most nanomaterials are rapidly cleared, often within hours or even minutes, from the bloodstream upon administration1-4. A variety of approaches have been investigated over the years in an attempt to extend blood circulation time for nanoparticles. These include, but are not limited to, tuning the physicochemical properties of the nanoparticles such as size5-7, shape8-10, charge5, 11 and stiffness12-14, camouflaging with PEG, natural proteins and polysaccharides15-20, hitchhiking on white blood cell (WBC), red blood cell (RBC) and platelet membranes21-25, mimicking virus nanostructures26 and exploiting self-recognition molecules such as CD4727-29. While these approaches have demonstrated various degrees of success, each has its own set of limitations. New innovative strategies for discovering novel circulation-prolonging molecules are highly desirable and would greatly help in engineering long-circulating nanoparticles with improved theranostic performance. M13 bacteriophage are rod-shaped biological nanoparticles, self-assembled from protein and nuclei acid molecules and possessing a width of about 7 nm and a length of approximately 1 μm30. Bacteriophages have short serum half life, and both antibody-mediated inactivation and clearance through the reticuloendothelium system (RES) have been shown to be critically important for phage elimination31-33. Adhaya and co-workers developed long-circulating phage lambda particles in 1996, through iterative rounds of selection and amplification of phages in mutator strains34, and later demonstrated that a single amino acid substitution in a capsid protein was sufficient to achieve long blood circulation35. Peptide phage display is a powerful technology for 3


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selecting

defined

material-interacting,

peptide

sequences

with

cell-internalizing,

a

desired

tissue-targeting,

property,

such

organ-homing,

as and

barrier-penetrating36-39. In particular, in vivo phage display has been used successfully to obtain peptides with the capability of homing to tumor vasculature, crossing blood-brain barrier, and delivering protein drugs transdermally, among many novel applications40-42. Notably, Albumin-binding peptides have been identified via page display and used to prolong blood circulation for therapeutic agents43, 44. Wolff et al. used

T7

phage

display

to

isolate

peptides

that

protected

phage

from

complement-mediated inactivation through binding to serum proteins45. The extensive prior work has laid a strong foundation for the hypothesis that a peptide phage display library, with a repertoire of over 109, should contain peptide sequences capable of extending the blood circulation time for M13 phage nanoparticles. Moreover, such identified blood circulation-prolonging (BCP) peptides are likely to be effective for extending the blood circulation time for other engineered nanoparticles. In this report, we demonstrate the feasibility of this approach by identifying a series of BCP peptides capable of extending the maximum blood retention time for M13 phage from 48 hours to 144 hours. Intriguingly, the majority of the identified BCP peptides contained an RGD motif, which mediated phage interaction with platelets to achieve long circulation. We further demonstrated that the BCP1 peptide, upon fusion to human heavy-chain ferritin (HFn) (Scheme 1a), significantly extended blood retention time, tumor cell uptake and anti-cancer efficacy for doxorubicin encapsulated in the HFn nanocage, a highly promising nanoparticle carrier system46, 47 (Scheme 1b). Our results demonstrated an innovative yet simple strategy for discovering novel BCP peptides and may have important implications for nanomedicine.

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Scheme 1. Schematic Illustration of engineered HFn nanocage displaying the BCP1 peptide and encapsulating anti-cancer drug doxorubicin (a), with the BCP1 peptide enabling prolonged blood circulation and enhanced tumor uptake of doxorubicin after in vivo administration (b).

To identify phages with the ability of prolonged circulation, we injected 1 x 1011 phage particles (plague forming unit, or pfu) of Ph.D.-C7C phage library, which had a peptide repertoire of 109, into the tail vein of Sprague-Dawley rats. We found that 48 hours (h) was the maximum elapsed time, at which point active phages could still be recovered from the bloodstream. About 300 phages, recovered at 48 h post library injection, were amplified and used for the second round screening. Dramatically prolonged blood circulation time was observed for these phages as compared to the library phage, and about 500 phages recovered at 96 h post injection were amplified and subjected to the third round screening. Further significant increase in blood retention over the previous round was observed, as revealed by the number of recovered phage at 48 h post injection (Figure 1a). During the second round screening, we randomly picked 30 phages at 96 h and conducted sequencing. Intriguingly, 19 of them contained RGD within their displayed sequences, and 6 of these 5


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RGD-containing sequences repeated at least twice and up to 3 times (Table 1). In comparison, 22 of the 30 phage clones randomly selected from the third round screening at 120 h post injection contained the RGD motif, with 6 of the RGD-containing sequences repeating at least twice and up to 6 times (Table 1). In contrast, none of the 15 clones randomly selected from the original library contained RGD within their displayed sequences and no repeating sequence was recovered (Supporting Information Table S1), indicating that the enrichment of RGD-containing clones was highly unusual and strongly suggesting that the RGD motif was important for the circulation-prolonging activity.

Table 1. Phage clones and their displayed peptide sequences selected from the second and third round of phage display screening.

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We next compared the relative circulation-prolonging ability of the three clones, namely BCP1 (CNARGDMHC), BCP2 (CIVRGDNVC) and BCP6 (CVPRGDMHC) respectively, which repeated at least 4 times on the third round screening. We co-injected the same number of these three phages together with a control phage randomly picked from the library (SC, with a displayed sequence CNATLPHQC) and recovered phage from the bloodstream at 0 h (3 min) and 72 h post injection. 30 clones were picked randomly from each time point for sequencing. As shown in Figure 1b, the recovery frequency of the four phages were nearly the same at 0 h, but at 72 h, SC phage disappeared. The other three clones were observed at a similar ratio, 7


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with BCP1 exhibiting slightly higher recovery frequency. We thus focused on BCP1 for subsequent experiments. To confirm the circulation-prolonging capability of BCP1, we injected the same number of BCP1 and SC phage into different rats and analyzed phage titer in the bloodstream at various time points. SC phage exhibited a much more rapid decay than BCP1 phage and were completely eliminated in the blood at 48 h (Figure 1c), while BCP1 was still detected at 144 h. To further confirm the prolonged blood retention capability of BCP1, we took advantage of a mutant phage (REW, with a displayed sequence CTARSPWIC), which had a mutation in the LacZa gene and appeared as white plaques while BCP1 phage appeared blue in the culture plates containing 5-bromo-4-chloro-3-indolyl-

b-D-galactoside

(X-Gal)

and

isopropyl-b-D-thiogalactoside (IPTG). REW had the same blood retention profile as SC (data not shown). We injected REW and BCP1 phage, mixed in a 1:1 ratio, to the same group of rats and assessed the change in their titer in the blood as time went by. The BCP1/REW ratio increased to over 100 at 12 h and over 1000 by 36 h (Figure 1d). To put the circulation-prolonging capability of BCP1 peptide into perspective, we compared the blood retention profile of BCP1 phage with that of the ABP phage, an engineered M13 phage that displayed a short albumin-binding peptide identified by Damico et al. through phage display44. The ABP phage exhibited excellent blood retention for the first 12 hours after IV administration, but its clearance accelerated after 12 hours, and by 36 hours the level of the ABP phage was nearly the same as the SC control phage (Figure S1). Thus, the BCP1 peptide was superior to the 8


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albumin-binding peptide in the long-term circulation-prolonging capability. Collectively, these results demonstrated the utility of in vivo phage display for isolating phage nanoparticles exhibiting prolonged blood circulation.

Figure1. Characterization of the long-circulating property of BCP1. (a) Enrichment of long-circulating phage following in vivo phage display screening. 1 x 1011 pfu of phage library, first round isolates and second round isolates were injected into tail vein of rats, and phage titer in the whole blood was assessed 48 h later. Mean ± S.E.M., n = 3, **p < 0.01, ***p < 0.001) (b) Relative circulation-prolonging activity of the isolated clones. 1 x 1011 pfu for each of the three BCP phages and SC were mixed and injected into the tail vein of rats. 30 clones were

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picked randomly at 0 h (3 min) and 72 h post injection and their identities revealed by sequencing. n = 3. (c and d) Confirmation of the circulation-prolonging capability of BCP1. In (c), the same number (1 x 1011) of BCP1 and SC phage were separately injected into different rats with phage titer in the blood determined at the various times, while in (d) BCP1 and REW, mixed in a 1:1 ratio, were injected to the same group of rats and then assessed for their relative phage titer by white/blue plague assay. Mean ± S.E.M., n≥3, *p < 0.05, **p < 0.01, ***p < 0.001). (e) Schematic representation of BCP1 mutant phage construction and the sequence of the displayed peptide for the three mutants, with the mutated amino acids shown in red. (f and g) Loss of the long-circulating property for the mutant phages. In (f), the same number (1 x 1011) of BCP1, SC and three mutant phages were separately injected into different rats with phage titer in the blood determined at the various times, while in (g) the individual phage were mixed with REW in a 1:1 ratio, injected to the same group of rats and then assessed for their relative phage titer by white/blue plague assay. Mean ± S.E.M., n = 3, ***p < 0.001)

The unusual presence of RGD in the displayed sequence of most of the isolated phages prompted us to assess the role of this motif for the long-circulation property. Three mutant BCP1 phages were constructed: TB1, which displayed a scrambled sequence of BCP1; TB2, which had RGD mutated to AGA; and TB3, which retained the RGD motif but had the other four flanking amino acids mutated to Alanine (Figure 1e). All of the three mutations led to dramatic loss of the long-circulation activity (Figure 1f). In fact, TB1 and TB2 behaved almost the same as SC, with no or very few phage found in the bloodstream at 48 h, indicating near complete loss of circulation-prolonging ability. TB3 also exhibited significant, but not complete, loss of the prolonging activity. The blue/white plague assay, by mixing the same number of the individual mutant BCP1 phage with REW, revealed very similar results, with TB3 losing much of the prolonging activity while TB1 and TB2 exhibiting a near 10


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complete loss (Figure 1g). The above results demonstrated that the ability of BCP1 peptide to prolong the blood-circulation of phages is highly sequence-specific, and that the RGD motif is necessary but insufficient for the circulation-prolonging activity, as the surrounding amino acids also played an important role. How could a 9-amino acid peptide promote persistent circulation of BCP1 phage in the bloodstream? Phages can be recognized by natural antibodies and inactivated by the complement system in the plasma48. Indeed, a time-dependent inactivation of phages was observed following incubation with plasma at 37 ℃, and this phage-inactivating activity was heat labile as much less phage inactivation was observed when plasma was pre-incubated at 56 ℃ for 10 minutes, consistent with the known heat sensitivity of the complement system (Figure 2a). Cobra venom factor (CVF) and ethylenebis (oxyethylenenitrilo) tetraacetic acid (EGTA), two agents known to inactivate the complement system45, significantly decreased plasma-elicited phage inactivation, further supporting the conclusion that the complement system played an important role in the observed phage inactivation by the plasma (Figure S2). Importantly however, BCP1 and SC phage did not show any difference in their inactivation profile, regardless whether the plasma has been heat-treated or not. These results indicated that the displayed sequence of BCP1 did not confer any resistance to complement-mediated inactivation of phage. Prior experiments with germ-free animals

lacking

antibodies

against

phage

have

demonstrated

that

the

reticuloendothelium system (RES) is sufficient for, and highly effective in the rapid removal of administered phage from the circulatory system32, and the presence of CD47 molecule, or the SELF peptide derived from it, on the surface of nanoparticles was able to reduce phagocytosis of nanoparticles by macrophages29. We thus asked whether the BCP1 peptide had a similar effect on the phagocytosis of BCP1 phage by 11


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the macrophage. No difference was observed between the BCP1 and the SC phage, in terms of the number of phage particles phagocytosed by the bone marrow derived macrophage cells (Figure 2b) or THP-1 macrophage cell line (Figure S3). Thus, the BCP1 peptide does not impact complement-mediated phage inactivation or macrophage-mediated phage uptake.

Phages as foreign particles are cleared from the body by phagocytes that must also recognize and avoid clearance of “self” cells. It has been amply demonstrated that the clearance of foreign particles could be delayed when attached to peripheral blood cells (PBC) including red blood cells (RBC), white blood cells (WBC) and platelets (PLT)21-24,

49.

Indeed, SC phage pre-bound to PBC exhibited prolonged blood

circulation, and PBC-bound BCP1 phage had further enhanced blood retention, as compared to their respective phage in the unbound form (Figure 2c). Interestingly, both BCP1 and SC phage, when pre-bound to PBC, were more resistant to plasma-mediated inactivation (Figure S4). Thus, the obvious possibility is that the BCP1 peptide promotes blood retention of phages through interaction with PBC, thus protecting phages from both phagocytic clearance and complement-mediated inactivation. To test this, we assessed the relative distribution of phages in the plasma and PBC at various times after administration. An order of magnitude higher number of SC phage was seen in the plasma as compared to PBC at 0 h, but as time went on, this difference narrowed (Figure 2d). By 36 h the number of SC phage in the PBC was actually slightly higher than in the plasma, although the number of phage remaining in the bloodstream at this point was low. These results were in full agreement with the above data showing that the SC phage bound to PBC were more resistant to clearance than the free phage. Consistent with the phage number change in 12


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the whole blood (Figure 1c), essentially no SC phage was detected in either the plasma or PBC at 48 h and beyond. A similar early distribution profile was observed with BCP1 phage, revealing nearly 10 times of more phage in the plasma than PBC at 0 h and about equal number of phage in the plasma and PBC by 24 h. Importantly however, there were significantly more phage in the PBC than in the plasma at 36 h and beyond, with the difference widening to over two orders of magnitude by 72 h (Figure 2e). These results strongly suggested that the circulation-prolonging property of BCP1 phage was due to their enhanced ability to bind to PBC. Furthermore, the TB2 phage, which differed from BCP1 only in the mutated RGD, reverted to a pattern analogous to SC phage, suggesting that the RGD motif was involved in the interaction of BCP1 with PBC (Figure 2f). To assess which component of the PBC was responsible for phage binding, we used a fractionation procedure to roughly separate PBC into RBC, WBC and PLT. Binding to PLT accounted for from 65% to over 90% of phage binding to PBC for BCP1 at the various time points (Figure 2g). Similar results were obtained for SC (Figure S5). To more accurately quantify phage binding, we used antibody-mediated FACS sorting to analyze the distribution of phages on the three major components of PBC following in vivo administration. BCP1 phage exhibited similar high-level binding to PLT and WBC but much lower binding to RBC, with significant binding still observed at 48 h and beyond, when assessed by the number of bound phage per million cells (Figure 2h). On the other hand, SC and TB2 phages showed relatively high binding to PLT, median binding to WBC and low binding to RBC at 0 h, but the binding decreased rapidly, with only low level binding detected to all three types of PBC at 24 h and beyond (Figure S6 and 7, respectively). Based on the published data on the abundance of the various PBC components in the rat blood50, we calculated the total number of phage bound to each of the PBC type. 13


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Consistent with the crude fractionation assay described above, PLT were found to account for the vast majority of the phage binding to PBC, while RBC and WBC contributed a small and negligible percentage of binding, respectively (Table 2). This is true for all three phages at different time points. Additional support for the critical importance of PLT binding came from in vitro studies. FACS sorting analysis following in vitro incubation of phages with the whole blood showed that WBC exhibited highest binding ability, PLT the median, and RBC the least, in terms of the number of phage bound per million cells (Figure 2i). Note that the detected number of bound phage per million cells was low for all the three blood components, as most of the phage presumably had been inactivated by the activated complement system following 90 min incubation as done for this experiment. Notably, BCP1 phage exhibited significantly higher binding ability than the SC phage to both WBC and PLT but not to RBC. But in terms of the overall number of bound phage, using the same calculation scheme as above, PLT was found to be the most important blood component, accounting for 84% of the total binding to PBC, while RBC and WBC contributed 10.29% and 5.71% respectively. The synthetic BCP1 peptide (ACNARGDMHCG; the flanking A and G were derived from the M13 coat protein and included to make the peptide more stable), but not the SC peptide (ACNATLPHQCG), significantly reduced BCP1 phage binding to PLT and WBC but not to RBC (Figure 2j). An IC50 of 373 μM was obtained for the BCP1 peptide to compete away the binding between the BCP1 phage and the PLT (Figure 2k), while no IC50 could be obtained for the SC peptide (Figure S8). In addition, no IC50 could be obtained for either the BCP1 or the SC peptide on the binding of SC phage to PLT (Figure S9 and S10). These results are consistent with the conclusion that BCP1 binding to PLT and WBC was mostly due to specific interaction mediated by the 14


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displayed peptide, while the binding of BCP1 phage to RBC was mostly due to non-specific adsorption. The BCP1 phage appeared to bind predominantly to the surface of PLT rather than being endocytosed, as over 91.7 % of the phage bound to PLT could be eluted following the glycine-HCl treatment (Figure S11). The RGD-mediated interaction also did not appear to affect the function of PLT, as the BCP1 phage did not cause PLT activation by itself, nor did it affect PLT activation elicited by ADP (Figure S12 and S13). Collectively, the above results demonstrated that the circulation-prolonging property of BCP1 phage was primarily due to specific interaction of phage with PLT, and also strongly suggested that the RGD motif mediated this interaction.

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Figure 2. BCP1 peptide promoted blood retention of phages through interaction with PLT. (a) Inactivation of phages by the plasma. BCP1 and SC phages (1 x 109) were incubated with plasma pre-treated at 37 ℃ or 56 ℃, and phage titer was determined at the various time points. Mean ± S.E.M., n = 3. (b) Phagocytosis of phages by BMDM cells in vitro was assessed at 37 ℃ by incubating 106 BCP1 and SC phages with BMDM cells respectively. Mean ± S.E.M., n = 3. (c) Prolonged blood circulation for PBC-bound phage. 1 x 108 pfu of phage BCP1 and SC, either unbound or pre-bound to PBC, were separately injected into rats and phage titer in the blood at the various time points were determined. Mean ± S.E.M., n = 3, **p < 0.01, ***p < 0.001, with BCP1 prebound compared to BCP1 while SC prebound compared to SC. (d-f) Relative distribution of phages in the plasma and 16


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PBC. 1 x 1011 of SC (d), BCP1 (e) and TB2 (f) were injected into rats through tail vein. Blood taken at different time points was centrifuged to separate plasma and PBC followed by phage titer determination. Mean ± S.E.M., n = 3, *p < 0.05, **p < 0.01, ***p < 0.001. (g) Assessment of the relative distribution of BCP1 in the three PBC components following crude fractionation. Blood was withdrawn at the various times after tail vein injection of 1 x 1011 of BCP1, separated into RBC, WBC and PLT by a crude fractionation procedure, and analyzed for phage titer. Mean ± S.E.M., n = 3. (h) Relative binding ability of BCP1 to the three PBC components in vivo. Blood was withdrawn at the various times after tail vein injection of 1 x 1011 of BCP1, separated into RBC, WBC and PLT by antibody-mediated FACS sorting, and analyzed for the number of bound phage per million cells/platelets. Mean ± S.E.M., n = 3. (i) Relative binding ability of BCP1 and SC to the three PBC components in vitro. 1 x 109 of BCP1 or SC were incubated with 1 ml of rat whole blood at 37 ℃ for 1.5 h, followed by antibody-mediated FACS sorting into RBC, WBC and PLT and determination of number of bound phage per million cells/platelets. Mean ± S.E.M., n = 3, ***p < 0.001. (j) Competition of BCP1 binding to PBC by peptides. 1 x 109 of BCP1 were incubated with 1 ml of rat whole blood at 37 ℃ for 1.5 h in the presence or absence of synthetic BCP and SC peptides (500 μg/ml), followed by antibody-mediated FACS sorting into RBC, WBC and PLT and determination of number of bound phage per million cells/platelets. Mean ± S.E.M., n = 3, **p < 0.01, ***p < 0.001. (k) Binding of BCP1 phage to PLT in the presence of increasing concentration of BCP1 peptide. PLT separated from whole blood were incubated with increasing concentrations of synthetic BCP1 peptide at 37 ℃ for 1.0 h, followed by incubation with phage (5x107) for 1.5 h. The number of bound phage was determined after centrifugation. The inhibition ratio was calculated according to the formula: (A-B) / A x 100. A: the number of bound phage in the absence of peptide; B: the number of bound phage pretreated with different concentrations of peptide. The value of IC50 was calculated by Graphpad software.

Table 2. Distribution of phages among PBC components and plasma.

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Shown is the number of phage associated with the various blood components in 1 ml of blood, with the relative percentage shown in parenthesis. Calculation was based on the number of phage per million blood cells/platelets (Figure 2h for BCP1 and Figure S6 and 7 for SC and TB2, respectively) and the number of blood cells/platelets per ml of blood, taken from Ref50. (Platelets: 1.14 x 109; WBC: 7.84 x 106; RBC: 7.01 x 109). PLM, plasma.

We next attempted to demonstrate the utility of the BCP1 peptide by creating BHFn, a hybrid protein formed by the insertion of the BCP1 peptide sequence into the N-terminus of human heavy-chain ferritin (HFn; Figure 3a). Both HFn and BHFn were expressed in E. coli and purified to >95% homogeneity (Figure S14a), with their identities confirmed by Western blotting using anti-ferritin antibody (Figure S14b). In agreement with published reports, HFn self-assembled into a spherical hollow nanocage architecture with an outer diameter of approximately 12 nm and an interior cavity of about 8 nm in diameter51, as revealed by transmission electron microscopy (TEM; Figure 3b). BHFn formed a similar but slightly larger nanocage, indicating that 18


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the addition of the BCP1 peptide sequence did not affect the self-assembly behavior of HFn. Largely consistent with TEM, dynamic light scattering (DLS) analysis revealed an average size of 13.5 nm for HFn and 14.3 nm for BHFn, respectively (Figure S14c and S14d). In agreement with the BCP1 phage results, BHFn did not elicit platelet activation (Figure S15). We used the well-adopted pH-facilitated denaturation/renaturation scheme to load doxorubicin (Dox) onto the ferritin nanocages52, and a loading factor of approximately 500 μg of Dox per μmol of protein was achieved for both HFn and BHFn. A time-dependent slow release of nanocage-loaded Dox was observed at pH 7.0, with less than 17% of Dox released over the 48 hour period, but Dox release was much accelerated at pH 5.0, with over 50% of Dox released for the first 10 hours and close to 80% released by 48 hour (Figure 3c). Dox release profile for HFn and BHFn was nearly identical. Compared to free Dox, HFn-loaded Dox (HFn-Dox) exhibited higher cytotoxicity towards B16 mouse melanoma cells (Figure 3d), a result that was to be expected as B16 melanoma cells are known to express transferrin receptor 1 (TfR1)53, the receptor for ferritin, and targeting through TfR1 would presumably lead to increased uptake of HFn-loaded Dox. Notably, BHFn-loaded Dox (BHFn-Dox) showed a further, statistically-significant increase in cytotoxicity over HFn-Dox, indicating that the BCP1 sequence, most likely the RGD motif, facilitates additional targeting towards the B16 melanoma cells and leads to further enhanced cancer cell killing. In full agreement, cytosolic Dox concentration measurement revealed that treatment with HFn-Dox resulted in increased Dox uptake in B16 melanoma cells over the free Dox treatment, but a further statistically-significant increase in Dox uptake was observed in the cells treated with BHFn-Dox (Figure 3e). This was true for both of the Dox concentrations tested. Fluorescent microscopy further confirmed 19


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these results (Figure S16). Similar to the situation in rats, BCP1 phage exhibited prolonged blood circulation in mice, albeit at slightly lower efficiency than in rats (Figure S17). To assess whether the BCP1 peptide could prolong blood circulation for nanocage-loaded Dox, we injected free Dox, HFn-Dox and BHFn-Dox into the tail vein of mice and measured Dox concentration in the whole blood at various times post injection (Figure 3f). Prolonged blood retention for HFn-Dox over free Dox was observed, in agreement with the published report47. Importantly, BHFn-Dox exhibited a further, statistically-significant improvement in the blood retention profile over HFn-Dox, as demonstrated by increased T1/2 (P

Blood circulation-prolonging peptides for engineered nanoparticles

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