Awakening p53 in vivo by D-peptides-functionalized ultra-small nanoparticles: Overcoming biological barriers to D-peptide drug delivery.

Peptides are a rapidly growing class of therapeutics with many advantages over conventional small molecule drugs. Dextrorotary (D)-peptides, with increased enzymatic stability and prolonged plasma half-life in comparison with natural L-peptides, are considered to have great potential as recognition molecules and therapeutic agents. However, the in vivo efficacy of current therapeutic D-peptides is hindered by their inefficient cellular uptake in diseased tissues.
Methods: To overcome physiological and cellular barriers to D-peptides, we designed a gold-based ultra-small nanocarrier coupled with polylysine (PLL) and a receptor-targeted peptide to deliver therapeutic D-peptides. Using a D-peptide p53 activator (DPA) as a proof of concept, we synthesized, functionalized and characterized gold- and DPA-based nanoparticles termed AuNP-DPA.
Results: AuNP-DPA were effectively enriched in tumor sites and subsequently internalized by cancer cells, thereby suppressing tumor growth via reactivating p53 signaling. More importantly, through a series of in vivo experiments, AuNP-DPA showed excellent biosafety without the common side effects that hinder p53 therapies in clinic trials.
Conclusion: The present study not only sheds light on the development of AuNP-DPA as a novel class of antitumor agents for drugging the p53 pathway in vivo, but also supplies a new strategy to use D-peptides as intracellular PPI inhibitors for cancer-targeted therapy.

Introduction

Protein-protein interactions (PPIs) mediate a great number of regulatory pathways and play a key role in many pathologies including tumorigenesis, and therefore represent an important and yet largely unexploited class of therapeutic targets 1, 2. However, compared to the generally well-defined active sites of enzymes, receptors and ion channels, the interaction surfaces of PPIs are always flat or moderately convex, which is much more challenging for traditional drug discovery and development efforts of small molecules 3, 4. Fortunately, peptides can overcome this obstacle due to their large interacting interface with chemical and structural diversity 5. Despite their obvious advantages over small molecules for targeting intramolecular PPIs, peptides suffer poor stability, making them unusable as drugs 6. In recent years, an array of techniques has been developed to improve the stability of peptides through chemical and/or structural strategies, thereby leading to a rapid expansion of the repertoire of peptide drug candidates for clinical evaluation. One of the most effective approaches to increase peptide stabilization is D-enantiomerization 5, 7.

D-peptides, composed entirely of unnatural D-amino acid residues, are resistant to proteolysis because the steric incompatibility of D-peptides and naturally occurring L-proteases affords an exceedingly high free energy barrier to catalysis 8. As a result, D-peptides are highly stable not only intracellularly but also in circulation 8, 9. An additional advantage of D-peptide therapeutics is that they are less immunogenic than L-forms because of their poor efficiency in proteasome-processed antigen presentation 10. Several D-peptide antagonists with antitumor activity have recently been identified using mirror image phage display techniques 11-14, promising a novel class of peptide therapeutics with clinical benefits.

Although the facile access to proteolytically stable D-peptide inhibitors makes it possible to target intracellular PPIs responsible for disease initiation and progression, there are still some major obstacles that need to be solved such as poor membrane permeability and low therapeutic target enrichment of D-peptides 15-17. Notwithstanding D-peptides can be introduced intracellularly for functional, mechanistic and efficacy studies via different delivery systems such as cell-penetrating peptides (CPPs) 18, 19 and liposomes 20. However, these delivery vehicles have serious drawbacks and disadvantages, such as rapid elimination by the liver and spleen, non-specific cellular uptake by normal tissues and organs, and toxicity, thereby preventing them from becoming therapeutically viable 21-23. Thus, there is a compelling need to develop new strategies for efficient intracellular delivery of D-peptides for clinical use.

Hitherto, aurous nanoparticle (AuNP)-based nanomedicine have emerged as an important strategy to deliver various payloads (such as small molecules, peptides and RNAs) into cells and target intracellular molecules 24-26. Moreover, AuNPs have irreplaceable advantages such as essential chemical inertness, non-toxicity, simple design and economic cost 24, 27. Thus, AuNP-based therapies have tremendous potential for clinical application, and some of them have entered clinical trials, such as CYT-6091 (complete phase 1 of clinical trials), AuroLase TM (complete early clinical trials), C19-A3 GNP (entered phase 1 of clinical trials) and so on 28, 29. Considering the specific tumor accumulation by enhanced permeability and retention (EPR) effect and intracellular-redox-triggered drug release by thiol-gold conjugation, AuNP-based drug delivery systems are particularly attractive for the treatment of solid tumors 30-32. In addition, receptor-ligand modification can promote transportation of nanoparticles carrying therapeutic molecules into cells through receptor-mediated endocytosis 25, 33, 34. Altogether, we hypothesize that receptor-ligand-modified AuNPs may be used as efficient carriers to deliver D-peptides for cancer therapy.

p53, a tumor suppressor that inhibits growth and induces apoptosis in response to cellular stress, plays a key role in preventing injured cells from becoming cancerous 35, 36. Dysfunction of p53 occurs in many human cancers, in which wild-type p53 is functionally inhibited by MDM2 37-39. Mounting evidence has shown that inhibiting MDM2-p53 interactions is a viable strategy for cancer therapy, and some small molecule antagonists have entered into clinical trials 40, 41. However, without exception, off-target toxicity poses a big challenge to the application of MDM2 antagonists, for which strict dose constraints has resulted in sluggish development and clinical application 42. Therefore, there is a compelling need to develop new p53 therapeutics with low nonspecific toxicity.

In this work, to overcome the biological obstacles of D-peptide application and develop new potent and safe p53 therapeutics, we investigated ultra-small AuNPs (<10 nm) as a high-loading drug delivery vehicle conjugated with D-peptide p53 activator DPA (TAWYANFEALLR) 20 for cancer therapy. Meanwhile, an RGD-derived peptide termed RGDDP (CRGDKRGDSP) was used to modify AuNPs in order to enhance their tumor targeting through specific binding to integrins, which have been demonstrated to be highly expressed on cancer cells but not on normal and stem cells 43-45. Our data indicate that AuNP-DPA may be used as a potential D-peptide-based therapeutic agent for cancer therapy.

Results and Discussion

Synthesis and characterization of AuNP-DPA

The preparation of ultra-small AuNPs-DPA is outlined in Figure . The Au core is an ultra-small gold nanoparticle, which was synthesized using the 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) procedure. In detail, 1 mL 10 mM HAuCl4 solution was mixed with 9 mL HEPES buffer (50 mM, pH 7.4) under vigorous stirring. After ~5 min, the solution color changed to red, indicating successful formation of the Au core. Transmission electron microscopy (TEM) (Figure ) and dynamic light scattering (DLS) measurements (Table ) of the Au core showed a 4 nm particle diameter and 5 nm hydrated particle size (PDI: 0.02). For Au core conjugation, an extra cysteine was added to the C terminus of DPA (DPA-Cys: TAWYANFEALLRC; Figure ), and the amino group at the N terminal was acetylated. Au-DPA was then obtained after 20 min vigorous stirring after adding 2 mL DPA-Cys solution (1 mg/mL in 50% acetonitrile and 50% PBS buffer) into the 10 mL Au core solution, following ultrafiltration (cut off: 10000 Da) and cleaning with the PBS 3 times to remove unconjugated DPA. Next, Fourier transform infrared (FTIR) spectroscopy confirmed conjugation of DPA to the Au core by the characteristic peaks of peptide at 3400 and 1650 cm-1 (Figure ). Notably, to investigate the effect of particle size on DPA loading, four Au cores with hydrated particle sizes of ~5 nm (PDI: 0.02), 10 nm (PDI: 0.05), 20 nm (PDI: 0.06) and 40 nm (PDI: 0.10) were prepared and used to load DPA. As shown in Table , the smallest Au core (ultra-small gold nanocore) had the maximum drug loading, which is mainly because this Au core has the maximum specific surface area.

To endow the ultra-small nanoparticles with better hydrophilicity and more functional groups to link with targeting molecules, polylysine (PLL) was used to encapsulate Au-DPA (Figure ). Considering that that the Au core was formed by the HEPES method and the pKa values for HEPES are pKa,1 = 3 and pKa,2 = 7.5, to guarantee sufficient deprotonation of PLL, pH 8.0 was chosen to promote PLL binding to Au-DPA. In this case, a negatively charged Au-DPA surface is also suitable for PLL electrostatic attachment. After this process, the intergranular electrostatic repulsion from the PLL-coated nanoparticle should prevail over the weak Van der Waals forces toward aggregation, thereby increasing the stability of the gold colloid 46, 47. The Zeta potential confirmed PLL binding as evidenced by a surface charge increase from -34.0 mV for Au-DPA to 27.8 mV for Au-DPA-PLL (Figure ). In addition, compared to the Au core, the size of Au-DPA-PLL increased, as shown by TEM (Figure ) and DLS (Table ), further proving the PLL and DPA conjugation.

With an aim of take full advantage of active targeting in vivo, integrin was selectively targeted to achieve strong tumor targeting and enhance delivery of the therapeutic molecules. Towards this end, an RGD-derived peptide termed RGDDP (RGDSPRGDR; Figure ) was used to modify Au-DPA-PLL. The large number of amino groups in PLL supplies sufficient sites for peptide conjugation. The reaction between the carboxyl of the C-terminal of RGDDP and the amino groups of PLL was catalyzed by EDC and NHS, both of which helped in the formation of more amide bonds by protecting them from hydrolysis 26, 48. Next, the final product named AuNP-DPA was collected by centrifugation and lyophilization. Of note, no aggregation was observed during all of these processes.

AuNP-DPA were firstly characterized by Zeta potential measurements (Figure ), UV-vis spectroscopy (Figure ), FTIR spectroscopy (Figure ), TEM (Figure ) and DLS (Figure ). In brief, after RGDDP conjugation, the charge shifted from 27.8 mV (Au-DPA-PLL) to 30.6 mV (AuNP-DPA), indicating successful modification with RGDDP (Figure ). The characteristic IR absorbance of peptide (amido bond: ~3400 and 1650 cm-1) and PLL (free amino group: ~3000 cm-1) in FTIR spectroscopy further confirmed the successful synthesis of AuNP-DPA (Figure ). In addition, we found that the surface plasmon peak band of AuNP-PDA was indistinct in the UV-vis absorbance measurement, probably due to the small Au core size and PLL coating (Figure ). TEM analysis revealed that AuNP-DPA was ~5 nm, spherical and had a uniform dispersion (Figure ), which was consistent with the DLS results (Figure ). Moreover, AuNp-PDA was very easily soluble in water and maintained monodispersity after 24 h incubation in 10% fetal bovine serum (FBS) in PBS buffer at pH 7.4 (Figure ), indicating that AuNP-PDA is stable under serum condition. Additionally, to determine the peptide loading, AuNP-PDA was dissolved in a test buffer containing 6 M GuHCl (to eliminate the interaction between DPA and PLL) and 1 M DTT (to break the conjugation between peptide and Au nanoparticle). The amount of released peptide was quantified by HPLC and calculated by the standard curve method, and the loading of DPA-Cys was found to be 0.5 mmol/g (mmol/g = peptide molarity/Au mass) (Figure ).

AuNP-DPA is efficiently internalized by cancer cells via integrin-dependent micropinocytosis

In general, high cell internalization and stimuli-responsive cargo release are required for delivering D-peptide into cancer cells and to awake the function of the therapeutic peptides. Based on our design, we consider that the positive charge from PLL and RGDDP conjugation can endow AuNP-DPA with good ability to be internalized by cancer cells. Thus, to determine this, we firstly treated an integrin-positive cell line HCT116 with AuNP-DPA (20 μg/mL) and its RGDDP-deleted counterpart (20 μg/mL) for 1 h, 2 h, 4 h and 6 h, and performed laser scanning confocal microscopy (LSCM) to detect their cellular uptake (Figure ). After 6 h incubation, bright green fluorescence from FITC-labeled DPA was found in the AuNP-DPA-treated cells, whereas RGDDP-deleted counterpart-treated cells presented relatively weaker fluorescence at the same exposure time (Figure ). Notably, free DPA and AuNP-DPA without PLL and RGDDP showed nearly no cellular uptake (Figure ), suggesting that cellular internalization of AuNP-DPA stems from the positive charge and RGDDP conjugation. To further clarify the advantages of the PLL coating and RGDDP modification, we quantified the fluorescence intensity of the LSCM images (Figure ), and the results showed that the increase in fluorescence intensity from intracellular uptake of AuNP-DPAFITC was significantly faster than that of AuNP-DPAFITC without RGDDP (Figure ). In addition, the average fluorescence intensity of AuNP-DPAFITC-treated cells was ~4-fold higher than that of cells treated by the RGDDP-deleted counterpart, and ~8-fold higher than that of cells treated by the RGDDP & PLL-deleted counterpart (Figure ). This result was also supported by flow cytometry, in which AuNP-DPAFITC was shown to be taken up by the cells up to 98.5%, whereas the cellular uptakes of RGDDP-deleted counterpart and RGDDP & PLL-deleted counterpart were only 60.1% and 10%, respectively (Figure ). Collectively, our data indicate that PLL-coated and RGDDP-modified gold nanoparticles can be taken up by cancer cells.

To explore the cellular uptake pathway of AuNP-DPA, free RGDDP and Amiloride (a specific inhibitor of micropinocytosis) were used to block internalization. By this way, we found that the cellular uptake of AuNP-DPA was completely inhibited after RGDDP or amiloride preincubation (Figure ), indicating that the cellular uptake was very likely contributed by micropinocytosis. Additionally, as micropinocytosis is actin-dependent, the cellular uptake of AuNP-DPA was also sufficiently inhibited by the actin inhibitor cytochalasin D (Cyto D) at a nontoxic concentration (Figure ). Collectively, these findings demonstrate that AuNP-DPA internalizes into cancer cells via integrin-dependent micropinocytosis.

Next, we investigated the intracellular distribution of AuNP-DPA following micropinocytosis. To this end, HCT116 cells were incubated with AuNP-DPA (20 μg/mL) for 6 h and then dyed with known markers for early endosomes (EEA1), late endosomes, and lysosomes (Lysotracker). As shown in Figure , the image of red-dye-labeled subcellular organelles and FITC-labeled nanoparticle presented that AuNP-DPA did not colocalize to late endosomes and lysosomes, but there was some overlap with early endosomes. These results demonstrate that AuNP-DPA can escape from early endosomes, which effectively avoids sequestration and degradation of nanoparticle or cargo in lysosomes.

AuNP-DPA releases DPA triggered by the reductive intracellular environment

The therapeutic efficacy of AuNP-DPA depends on efficient release of DPA to the cytosol. This process can be triggered by an intracellular reductant such as GSH to break the gold-thiolate bonds, as shown in Figure . To test this, we incubated AuNP-DPA (~0.5 M, DPA) in PBS without GSH for two weeks and then, after centrifugation, the supernatant was detected by HPLC.

No DPA peptide was found in the supernatant, suggesting that the gold-thiolate bond is highly stable, thereby prolonging the in vivo circulation half-life of AuNP-DPA (Figure ). In sharp contrast, DPA-Cys peptides were detected and quantitatively recovered after 4 h incubation with intracellular redox environment mimic solution (10 mM GSH in PBS buffer, pH 7.4) (Figure ). Next, HPLC was used to monitor the release kinetics of DPA-Cys from AuNP-DPA. Incubation in redox environment mimic solution resulted in ∼50% cumulative release by ~0.6 h (Figure ) and ∼100% release by 2 h (Figure ), indicating that conjugated DPA-Cys may be efficiently released by intracellular reducing environments.

AuNP-DPA inhibits cancer cell growth in vitro in a p53-dependent manner

It is well known that approximately one-third of all human cancers suffer from p53 dysfunction 38, 49, 50. Previous reports found that DPA reactivates p53, which, in turn, induces cell cycle arrest and apoptosis 20. To verify this, we treated isogenic HCT116 colon cancer cells carrying intact p53 (HCT116 p53+/+) or lacking p53 (HCT116 p53-/-) with 2 μM AuNP-DPA, 2 μM free DPA, 4 μg/mL AuNP and 2 μM Nutlin3. Of these treatment groups, ~4 μg/mL AuNP, equivalent in weight to 2 μM AuNP-DPA, was used as a negative control, and 2 μM Nutlin3, a small -molecular activator of p53 51, served as positive control. 72 h after treatment, AuNP-DPA and Nutlin3 significantly induced apoptosis of HCT116 p53+/+ cells, whereas they had almost no effect on the apoptosis of HCT116 p53-/- cells (Figure and Figure ). To confirm that the delivered PMI inhibits p53-MDM2/MDMX interaction, we explored the expression of p53 and p21 in HCT116 cells by western blot (Figure ). As shown in the quantitative data from three independent experiments in Figure , after 24 h treatment, AuNP-DPA and Nutlin3 increased the expression of p53 and its downstream gene p21 52, 53 significantly. All of these results suggest that DPA inhibited cancer cell proliferation in a p53-dependent manner. In addition, we also evaluated the effect of AuNP-DPA on cell cycle distributions of HCT116 p53+/+ and HCT116 p53-/- cells. The results showed that AuNP-DPA and Nutlin3 significantly induced cycle arrest of HCT116 p53+/+ cells at G0/G1 phase in comparation to PDA and AuNP (Figure ), whereas treatment with AuNP-DPA and Nutlin3 almost did not affect the cell cycle distributions of HCT116 p53-/- cells (Figure ). These findings further support that AuNP-DPA inhibits cancer cell growth in a p53-dependent manner.

AuNP-DPA specifically enriches in the tumor sites

Based on our design combining the inherent EPR effect with integrin targeting, AuNP-DPA can specifically accumulate in the tumor site. The EPR effect is a phenomenon observed in solid tumors where excessive angiogenic signals result in the formation of defective 'leaky' tumor vasculature, through which nanoparticles (NP) <150 nm in size can extravasate to the tumor microenvironment 54. Additionally, the increased tumor volume also leads to ineffective lymphatic drainage, further promoting NP retention 55. In addition to the EPR effect, actively targeted delivery of antitumor drugs/molecules to cancer cells is an optimized and effective strategy to improve the efficacy of anticancer therapies 56. Nanoparticle selectivity can be improved by specifically binding to proteins specifically upregulated in cancer cells such as integrins 57, 58. In this context, RGDDP, consisting of two integrin-binding motifs RGDK 43 and RGDSP 44, was linked to the nanoparticles to further improve tumor targeting.

Long blood circulation time is an important requirement for in vivo target-specific drug delivery 59. We explored the circulation time of AuNP-DPA with an injection dose of 2 mg/kg in mice, and the nanoparticle presented a long half-life of 7.5 h with a first order elimination rate (Figure ) 60. To assess tumor targeting of AuNP-DPA, the bio-distributions of AuNP-DPA and its counterpart without RGDDP were examined in normal organs and tumor sites. Toward this end, a red fluorescent dye, sulforhodamine 101 acid chloride, was first conjugated to the amino terminal of DPA. The semi-quantitative results showed that AuNP-DPA exhibited higher accumulation in the tumor sites than its counterpart without RGDDP at 6 h after intravenous injection (iv) (Figure ) or intraperitoneal injection (ip) (Figure ). Through further analysis of the fluorescence distribution, either via ip or iv, we found AuNP-DPA expectedly showed 2-fold greater tumor fluorescence than its RGDDP-deleted counterpart (Figure ), suggesting that RGDDP further increased enrichment of the nanoparticles in the tumor sites. In addition, all tumor-to-organ ratios for AuNP-DPA were significantly higher than those for its RGDDP-deleted counterpart (Figure ). Taken together, these findings indicate that integrin targeting can improve the tumor selectivity of AuNP-DPA.

Potent antitumor activity of AuNP-DPA in HCT116 p53+/+ xenograft model

To explore the anti-cancer activity of AuNP-DPA in vivo, 25 mice xenografting HCT116 p53+/+ tumors (50-100 mm3) were randomly and equally divided into five groups: AuNP-DPA, AuNP (the DPA-deleted counterpart of AuNP-DPA), free DPA, doxorubicin (positive control) and PBS (control). Given that there exists no difference in the bio-distributions of AuNP-DPA administered iv or ip (Figure ), user-friendly ip administration was adopted and the mice were treated every other day at a dosage of 2 mg/kg for 13 days. The study design is illustrated in Figure . Of note, there are two irresistible reasons for us to choose doxorubicin (DOX) as a positive control in this experiment: (1) DOX is a first-line chemotherapy and has been used for a long time, whereas Nutlin3 is only at the clinic trial stage; and (2) DOX treatment was more effective than Nutlin3 at the same dose toward this model (Figure ). After 13 days of treatment, free DPA peptide and AuNP displayed hardly any tumor-inhibitory effect (Figure and Figure ). In a sharp contrast, AuNP-DPA inhibited tumor growth by 88% on day 13 compared to the control, even better than DOX (inhibition ratio of 64.3% in comparison with the control), also shown in Figure and Figure .

At the end of the experiments, the xenograft tumors were isolated and weighed. As expected, the tumor in the AuNP-DPA-treated mice were much smaller than those in the AuNP-, DPA- or PBS- treated mice (Figure ). Notably, the average tumor weight of AuNP-treated mice was ~6-fold larger than that of AuNP-DPA-treated mice, suggesting that the main therapeutic effect is due to the anticancer activity of DPA rather than nonspecific toxicity from the nanoparticles. Meanwhile, the geometric mean tumor weight of the AuNP-DPA-treated mice (26.4 mg) was just 12% that of the free DPA-treated mice (221.2 mg, P < 0.01), indicating that this nanosystem awakens the function of DPA.

To further verify the antitumor activity of AuNP-DPA at a pathological level, we first examined the above xenograft tumor tissues with H&E staining. As shown in Figure , morphological integrity was observed in the PBS-, DPA-, and AuNP-treated groups. Conversely, we observed apoptotic or necrotic tumor tissues in the DOX- and AuNP-DPA-treated mice, particularly in the latter. Next, TUNEL assay was performed for the evaluation of the extent of apoptosis. Expectably, a crowd of TUNEL-positive (apoptotic) cells can be found in the DOX- and AuNP-DPA- tumors, whereas scarcely any apoptosis can be observed in the DPA- or AuNP- treated samples (Figure ). Collectively, our results provide an abundance of evidence that AuNP-DPA possessed potent in vivo anticancer efficacy even better than the first-line chemotherapy drug DOX.

AuNP-DPA inhibits tumor growth in vivo through reactivating the p53 signaling pathway

The tumor suppressor protein p53 plays a critical role in maintaining genomic stability as well as inhibiting malignant proliferation, and impairment of p53 signaling is a hallmark of cancers 42, 61. There is evidence demonstrating MDM2 and/or MDMX antagonism by small molecules or peptides is a clinical strategy for cancer therapy 42. The DPA used in the present study is a D-enantiomeric peptide with high affinity for both MDM2 and MDMX 20. Thus, AuNP-DPA should exert its antitumor effect through reactivating the function of p53 via releasing p53 from the MDM2 (MDMX)/p53 complex (Figure ). To validate this, immunohistochemistry staining of p53, p21 and Ki67 were observed in all tumors with serial sections. In line with the in vitro WB results, p53 and p21 were apparently up-regulated in the AuNP-DPA-treated tumors compared to PBS-treated tumors, whereas DPA or AuNP treatment almost did not affect the levels of p53 and p21 relative to those of the control (Figure ). This was consistent with the TUNEL results shown in Figure . In addition, all of the PBS-, DPA- or AuNP- treated tumors showed extensive positive staining of the proliferation marker- Ki67, whereas the number of Ki67-positive cells decreased significantly in the AuNP-DPA-treated tumor (Figure ). Collectively, our data indicate that AuNP-DPA inhibits tumor growth in vivo via reactivating p53 signaling.

Safety evaluation of AuNP-DPA

As mentioned above, MDM2 and/or MDMX antagonism could be an effective strategy for cancer therapy through reactivating p53 pathway 49, 62. Thus, in recent years, a number of selective small molecule MDM2/MDMX inhibitors have been widely developed including a class of imidazoline compounds termed nutlins, several of which have entered in clinical trials 4, 40. Nutlins have been demonstrated to effectively kill B-cell chronic lymphocytic leukemia (B-CLL) cells; however, they also induced some toxicities to normal cells: such as B lymphocytes and hematopoietic stem cell 63. What's worse, in a clinical trial of nutlin derivative, termed RG7112, almost all patients endured at least one drug-related side effects, including but not limited to thrombocytopenia, leukopenia and neutropenia. 64. In addition, it has been reported that nutlins can directly cause DNA damage of the normal cells and indirectly promote stem cell differentiation 40-42. Thus, there is a compelling need to develop new therapeutics with lower nonspecific toxicity.

To overcome the systemic toxicity from off-targeting, a feasible route is to load the anti-cancer drug in a tumor-specific vehicle 65, 66. To minimize side effects, a two-stage targeting strategy was designed in AuNP-DPA (Figure ). Benefited from the EPR effect (first targeting) and RGDDP conjugation (second targeting), AuNp-DPA can accumulate at the tumor sites specifically, resulting in minimized off-target effects and subsequent side effects. Moreover, DPA has strong specificity towards MDM2, which is specifically overexpressed in cancer cells. AuNp-DPA, as expected, had no effect on normal cells and a p53-null cancer cell line SW480, whereas DOX showed potent cytotoxicity. (Figure ).

To verify the in vivo biosafety of AuNP-DPA, comprehensive medical examinations were performed during and after anticancer treatment. After 13 days of administration, the body weights of DOX-treated mice significantly decreased, whereas no significant weight loss was found in other groups (Figure ). This was supported by several previous studies that DOX always suffered from some undesirable off-target effects 67, 68. Notably, except for DOX-treated mice, all other mice almost did not (each group respectively compared to control group by t-test, p>0.05) show significant haematological toxicities (Figure ), suggesting a relatively low toxicity of AuNP-DPA.

Given that liver and kidney are almost entirely responsible for drug biotransformation and elimination, we comprehensive assessed the health of these two organs. As shown in Figure , AuNp or DPA treatment almost did not affect the liver weight of mice compared to the control (t-test, p>0.05), whereas DOX-treated mice presented a statistically significant loss of liver weight relative to control mice (t-test, p<0.05). In addition, the levels of ALT and AST maintain normality in AuNP-DPA-treated mice (t-test, p>0.05) but increased significantly in DOX-treated mice (t-test, p<0.05), suggesting that DOX leaded to the abnormal hepatic function (Figure ). This result proved again by H&E staining: DOX-treated mice presented medium, spotty necrosis of the liver (Figure ). Next, kidney condition was evaluated by H&E staining (Figure ), CRE (Figure ) and BUN (Figure ). Similarly, mice treated by DOX showed increased levels of CRE and BUN and symptoms of glomerular lesions compared to control mice, whereas the other treatments had no harmful effects on the kidneys (Figure ). Moreover, mice treated by DOX showed spleen damage compared to control mice, while the spleen in other groups stay healthy (Figure ). It was observed that all treatments in this work, including DOX, didn't presented any other toxicities, for instance, cardiac trauma (Figure ) or allergic lung resistance (Figure ). Overall, AuNP-DPA is safe enough for future clinical use.

Conclusions

Redox-responsive delivery is a robust platform in nanomedicine for the development of peptide-derived anticancer therapeutics. The present study developed a gold-based nanocarrier decorated with a D-enantiomeric peptide coupled with PLL and receptor-targeted peptide (termed AuNP-DPA) for targeted tumor therapy in vivo. Profited by the EPR effect and RGD targeting, AuNP-DPA can successfully deliver DPA into cancer cells and specifically accumulate at tumor sites. Through a series of in vitro and in vivo experiments, we demonstrated that AuNP-DPA has excellent biosafety and potent antitumor activity. In summary, the ultra-small gold nanoparticles functionalized with therapeutic and targeting peptides may be a promising anticancer nanosystem to overcome physiological and cellular barriers for targeted delivery of therapeutic D-peptides and further awaken their antitumor efficacy.

Methods

Materials

HAuCl4·xH2O was purchased from Aladdin Chemicals. All synthetic peptides were obtained from CS Bio in Shanghai, China. We purchased chemicals from Sigma-Aldrich unless otherwise specified. Acetonitrile and water (HPLC grade) were purchased from Fisher Scientific Ltd. All products were used as received without further purification.

Fabrication of Au core, Au-DPA, Au-DPA-PLL and AuNP-DPA

An aqueous solution of tetrachloroauric acid (HAuCl4·xH2O, 1 mL, 10 mM) was mixed with 9 mL HEPES buffer (pH 7.4, 50 mM). After 10 min magnetic stirring, the solution color changed from golden yellow to wine red, which is the Au core. After this, 2 mg DPS-Cys was added into the Au core solution with stirring. Then, the color changed to bluish violet, indicating that DPA-Cys was successfully conjugated to the Au-core to form Au-DPA. Next, 1 mg PLL (MW 1500-2000) was added into the mixture, followed by a 10-min ultrasonic dissolution. The Au-DPA-PLL and its RGDDP-deleted counterpart (termed AuNP-DPA W/O-RGDDP) were collected by 14000 ×g centrifugation and lyophilization. To prepare AuNp-DPA, carboxylates (-COOH) of RGDDP-Cys were firstly reacted to a bifunctional linker N-succinimidyl maleimidoacetate. In detail, 0.5 mg RGDDP-Cys was dissolved in 1 mL DMF containing 0.5 mM N-succinimidyl maleimidoacetate. After 20-min reaction at room temperature, 1 mL DMF mixture was added dropwise into the 10 mL Au-DPA-PLL solution (1 mg/mL in PBS). After 30 min of stirring, the excess reactants were removed by dialysis (cutoff, 10 kDa) and washed three times with PBS buffer. AuNP-DPA were then freeze-dried for subsequent experiments.

Physicochemical properties of AuNP-DPA

TEM images were taken by an HT7700 operated at 100 kV acceleration voltages, to explore the morphology and lattice structure of the nanomaterials. DLS performed by Malvern Zetasizer Nano ZS system, was used to obtain the hydrodynamic size distribution of nanoparticle solutions (1 mg/mL in PBS, 1 mL). For Zeta potential measurement, the nanoparticles were solved in PBS at the concentration of 1 mg/mL, and measured at 37 °C. The FTIR was obtained by Nicolet 6700 via measuring the KBr-included freeze-dried sample powder. The UV-vis absorption spectroscopy was measured by Shimadzu 3000 spectrophotometer, at a concentration of 0.2mg/ml.

Quantification of drug loading and GSH-responsive drug release

To quantify drug loading, we firstly solve 6 M GuHCL and 1 M DTT into PBS buffer (pH 7.4). AuNP-DPA were dissolved in the prepared solvent, and the amount of released peptide was quantified by HPLC. To test GSH-responsive drug release, AuNP-DPA were dissolved in PBS buffer (pH 7.4) containing 10 mM GSH, and the nanoparticles were then removed by 14000 ×g centrifugation. Following this, the supernatants were quantified by HPLC and authenticated by ESI-MASS.

Cellular uptake

Cellular uptake of AuNP-DPA was detected by CLSM (FV1200, Olympus) and flow cytometry (BD Biosciences, NJ). FITC was firstly labeled to the N-terminal of DPA. Similarly, AuNp-DPAFITC and AuNp-DPAFITC W/O-RGDDP were prepared as above. We prepared solutions of DPAFITC, AuNp-DPAFITC and AuNp-DPAFITC W/O-RGDDP in culture medium at a concentration of 200 μg/mL. HCT116 cells were first cultured for 24 h. The medium was then replaced with the medium containing AuNp-DPAFITC or AuNp-DPAFITC W/O-RGDDP at pH 7.4, following 6h incubation at 37 °C. After washing the cells with PBS twice, cell imaging or flow cytometry analysis were carried out. DAPI (Molecular probes) was used to mark cell nucleus. Paraformaldehyde was used to fix the cells before observation. With regard to CLSM, the excitation wavelengths were 405 nm (3.15 mW) and 543 nm (0.7 mW).

Ethics statement

All animal procedures were approved by the Ethics Committee of Xi'an Jiaotong University, and performed following Institution Guidelines of Laboratory Animal Center of Xi'an Jiaotong University.

In vivo bio-distribution

Sulforhodamine 101 acid chloride-labelled DPA-Cys was firstly prepared to synthesize fluorescent AuNp-DPA or AuNp-DPA W/O-RGDDP. In detail, Texas Red™-X, Succinimidyl Ester purchased from Thermo Fisher was incubated with the N-terminal-free DPA-Cys in PBS buffer for 2 h and then purified by HPLC. From this, fluorescent DPA-Cys was obtained, and was used to synthesize fluorescent AuNp-DPA or AuNp-DPA W/O-RGDDP as in the above protocol.

Tumor cells (HCT116 p53+/+, 4×106 cells/site) were injected subcutaneously into BALB/c nude mice with four to five weeks old. After three to four weeks, each tumor-bearing mouse was injected with 200 μL fluorescent AuNp-DPA or AuNp-DPA W/O-RGDDP (2 mg/kg). Mice were humanely sacrificed at each indicated time, and the organ and tumor were immediately collected. IVIS Spectrum System was then used to semi-quantitatively explore the biodistribution of the nanoparticles (λex 590 nm; λem 610 nm).

In vivo circulation time

Pharmacokinetic studies were performed using the athymic BALB/c nude mice as In vivo bio-distribution. 200 μL Sulforhodamine 101 acid chloride-labelled AuNp-DPA (2 mg/kg) were administered by intravenous injection via tail vein. Approximately 100μL blood samples were collected from the retro-orbital sinus of anesthetized mice at indicated time points (n=3 at each time points) and reserved in heparin anticoagulation tube. The blood samples were determined using a microplate reader (λex 586 nm; λem 605 nm). The blood samples from PBS-administered mice as negative control, and these blood samples also were used to dilute the high-concentration AuNp-DPA as standard to quantify the blood samples from administered mice.

In vivo treatment response

HCT116 subcutaneous tumor models were established as described above. When all tumors grew up to 50-100 mm3, the mice were randomly divided into different groups (five mice per group). AuNP-DPA, AuNP and DPA wre administered at dose of 2 mg/kg on days 1, 3, 5, 7, 9, and 11. PBS solution was used as a negative control. Owing to the fact that (1) the amount of DPA in 2 mg/kg AuNP-DPA is approximately equivalent in weight to 1.5 mg/kg DPA, and (2) the dose over.

1.5 mg/kg would result in the drug-related death of BALB/c nude mice, the administration dose of DOX was 1.5 mg/kg every other day. Tumor volume was calculated as follow: volume = length × width2 / 2. Before H&E or immunohistochemical staining, the tumor and organ tissues were fixed and sliced into 5.0 μm sections. In addition, routine blood examination and Clinical biochemical indices were detected by the Clinical Laboratory of the First Affiliated Hospital of Xi'an Jiaotong University according to standard clinical laboratory procedures.

In vivo toxicity

To evaluate potential toxicities of AuNP-DPA, we monitored the body weight of all mice over the course of treatment and measured hematological indices as well as organ function indices after 13 days of treatment. Control mice were only implanted with xenograft tumor and did not receive any treatment. Forty-eight hours after the last dose, mice were anesthetized. We collected the blood to evaluate CBC including WBC, RBC, haemoglobin and platelet counts. Additionally, blood serum was used for ALT, AST, BUN and CRE assessment using ELISA kits. Animals were then euthanized according to the corresponding procedure. Prepared tissue slices were stained with H&E.

Statistical analysis

Statistical analyses were performed using two-sided Student's t-test. P < 0.05 was considered significant. Data are expressed as mean ± s.d. or s.e.

Supplementary methods about necessary synthetic procedure and cell experimental methods, figures S1-S19 and tables S1 and S2. Supplementary figures and tables.

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