Engineering peptide-targeted liposomal nanoparticles optimized for improved selectivity for HER2-positive breast cancer cells to achieve enhanced in vivo efficacy

Abstract

Here, we report rationally engineered peptide-targeted liposomal doxorubicin nanoparticles that have an enhanced selectivity for HER2-positive breast tumor cells with high purity, reproducibility, and precision in controlling stoichiometry of targeting peptides. To increase HER2-positive tumor cell selective drug delivery, we optimized the two most important design parameters, peptide density and linker length, via systematic evaluations of their effects on both in vitro cellular uptake and in vivo tumor accumulation and cellular uptake. The optimally designed nanoparticles were finally evaluated for their tumor inhibition efficacy using in vivo MMTV-neu transplantation mouse model. In vitro, we demonstrated that ~1% peptide density and EG8 linker were optimal parameters for targeted nanoparticle formulations to enhance HER2-positive cancer cellular uptake while preventing non-selectivity. In vivo results demonstrated that at 0.5% peptide density, enhancement of tumor cell uptake over non-targeted nanoparticles was ~2.7 fold and ~3.4 fold higher for targeted nanoparticles with EG8 and EG18 linker, respectively, while their accumulation levels at tumor tissue were similar to the non-targeted nanoparticles. These results were consistent with in vivo efficacy outcomes that ~90% tumor growth inhibition was achieved by Dox-loaded HER2 receptor targeted nanoparticles, TNP^HER2pep, over control while all nanoparticle formulations minimized overall systemic toxicity relative to free Dox. This study highlights the significance of understanding and optimizing the effects of liposomal nanoparticle design parameters for enhancement of tumor selectivity to achieve improved in vivo therapeutic outcomes.

1. Introduction

Breast cancer is the most frequently diagnosed and 2nd deadliest cancer for U.S. females [1]. Among the various types of breast cancer identified so far, human epidermal growth factor receptor 2 (HER2) stands out as a target of importance due to its cellular overexpression and poor prognosis in 25–30% of breast cancer patients [2,3]. Currently, trastuzumab, a HER2 targeted therapeutic antibody, either as a single agent or in combination with chemotherapeutic agents, is used as a standard-of-care treatment for HER2-positive (HER2+) breast cancer [4-6]. Despite improved clinical outcomes from the current standard treatments, these drugs induce toxic side effects on heart and lung tissues, such as interstitial pneumonitis and congestive heart failure, in a subset of patients [2,4-7].

To treat HER2+ breast cancer effectively with reduced toxicities, HER2 targeted nanoparticle (TNP) based drug delivery systems have been investigated using various targeting moieties, therapeutic agents, and nanocarriers over the past decade [8-17]. Specifically, anti-HER2 monoclonal antibody (mAb) or single-chain variable fragment (scFv) conjugated nanoparticles have been investigated in pre-clinical studies for efficacy in HER2+ breast cancers [8-17]. Although anti-HER2 mAb and scFv molecules have high specificity for the target HER2 receptors, they bind not only to HER2 receptors on target cancer cells, but also to HER2 receptors on healthy cells with regular HER2 expression levels, due to their high affinity for HER2 receptors. This specific yet non-selective binding to HER2 receptors on healthy cells reduces the concentration of drug that actually reaches the tumor while increasing the likelihood of unintentional toxic effects [8]. As a result, the high-affinity HER2 targeting approach is prone to fall short of achieving an optimal patient outcome [18-20].

To overcome non-selectivity attributed to a high affinity targeting approach, one strategy is to harness multivalency using multiple copies of a targeting moiety that has a weak-to-moderate affinity [18,21]. Different from the high affinity versions, weaker affinity moieties provide a reduced residence time for TNPs on the cellular surface owing to their shorter dissociation half-lives, resulting in a decreased probability of drug delivery to HER2 expressing healthy cells. Meanwhile, when the same TNPs encounter HER2 overexpressing cancer cells, they simultaneously form multiple binding interactions that result in enhanced avidity and in turn significantly extend residence time [18,21-25]. As a result, this targeting approach would preferentially deliver the drug only to those cells with overexpressed HER2 receptors on their surfaces, i.e., breast cancer cells, while sparing the healthy cells. Among targeting moieties, peptides are excellent candidates due to their small size, ease of synthesis, and lower immunogenicity, specificity, and, most important, moderate monovalent binding affinity to the target receptors as compared to the mAb and scFvs, while still preserving specificity [18,21,26,27].

We previously developed a peptide-targeted liposomal nanoparticle for HER2+ breast cancer cells that potentially could be further engineered to achieve selectivity in targeting [28-30]. Our research primarily evaluated how peptide valency and binding avidity of TNPs affected in vitro cellular binding and uptake for HER2+ breast cancer cell lines. Also, the length of the linker connecting the peptide to the nanoparticle significantly influenced the in vitro cellular uptake efficiency of HER2 TNPs. While shorter linkers improved the targeting efficiency of the peptides, longer linkers hindered target binding, due to the different lengths of ethylene glycol (EG), which adopt different structural conformations. Shorter repeating units of EG maintain a more linear conformation, while longer EG variants prefer a mushroom-like globular conformation [28-30]. Hence, although TNPs had the same peptide density, shorter linkers improved the accessibility of peptides to target receptors, achieving enhanced cellular binding and uptake in vitro.

On the other hand, using longer linkers generally increased the in vivo particle accumulation at the tumor site compared to shorter linkers [31]. This was accomplished by extending the in vivo half-life of the circulating particles by circumventing non-specific interactions with random cells and reticuloendothelial system (RES) clearance. Nevertheless, the higher particle accumulation from using longer linkers would not necessarily increase the particle tumor cell uptake. For instance, in the same in vivo study, we identified peptide densities for TNPs with shorter linkers that showed significant enhancements in tumor cell uptake, while their accumulation levels were similar to longer linkers at the given peptide densities [31]. Consequently, these results suggest a clear need for optimization both in vitro and in vivo of both TNP peptide density and EG linker length, since in vitro results seldom directly correlate to in vivo outcomes.

To date, despite the impact that both peptide density and linker length have on nanoparticle drug delivery, few studies have evaluated these parameters in vivo. The main objectives of this study were: 1) to investigate the impact of changes in the nanoparticle design parameters on efficacy and 2) to identify optimal TNP formulations with highest therapeutic efficacy for HER2+ breast cancer. As a targeting ligand, we utilized a cyclic HER2 binding peptide (HER2pep) that we evaluated in the earlier studies [28-30]. Doxorubicin (Dox) was selected as the chemotherapeutic agent, since it is a common choice in the treatment for HER2+ breast cancer [2,4]. Building upon our in vitro studies, we expanded our in vitro binding/uptake studies to identify the nanoparticle formulation that would deliver the best results in vivo. We further optimized in vivo HER2pep presenting targeted nanoparticles (TNP^HER2pep) by tissue biodistribution and tumor cell uptake studies in animal models. Finally, we evaluated in vivo antitumor efficacy and toxicity of Dox prodrug-loaded TNP^HER2pep. The results demonstrate that an enhanced efficacy with minimal systemic toxicity is achieved by in vitro and in vivo optimization of design parameters of Dox prodrug-loaded TNP^HER2pep as compared to non-targeted Dox prodrug-loaded nanoparticles (NP_Dox). This study highlights the importance of understanding relationships between valency and linker length for peptide-targeted nanoparticle drug delivery approach and introduces an integrative strategy for nanoparticle design to enhance tumor selectivity to accomplish improved therapeutic outcomes.

2. Results

2.1. Design and preparation of Dox prodrug-loaded TNP^HER2pep

To improve the nanoparticle drug delivery and efficacy in HER2+ breast cancer, we designed nanoparticles with optimized HER2 targeting peptide density and linker length that target and efficiently kill HER2+ breast cancer cells in vivo (Fig. 1A, B). We selected a cyclic peptide as the HER2 targeting moiety for this study (HER2pep), particularly because of its weak-to-moderate affinity (K_d of ~180 nM) [30]. We hypothesized that due to the short half-life of a single binding interaction between the peptide and HER2 receptor, residence time of a TNP on a healthy cell surface will be too short to exert its cytotoxic effect. In comparison, when a TNP encounters a cancer cell overexpressing HER2, it will be able to form multiple binding interactions simultaneously between multiple peptides on its surface and multiple HER2 receptors on the cancer cell, which in turn significantly increases its avidity and residence time. This preferential multivalent binding effect accomplishes selectivity in targeting by favoring higher binding avidity to cancer cells and selectivity in endocytosis of the drug-loaded nanoparticles (Fig. 1A). In contrast, TNPs synthesized with high affinity targeting moieties, such as anti-HER2 mAbs and scFv, will deliver the drug payload not merely to cancer cells but also to healthy cells with low HER2 expression levels because even a single high-affinity binding interaction will have a long half-life of dissociation.

Fig. 1.

Cartoon representation of selective targeting design strategy of Dox prodrug-loaded TNP^HER2pep for HER2 overexpressing breast cancer cells. (A) In the case of ~25–30% of breast cancer patients, expression of HER2 on epithelial cells within breast cancer legion is ~100–1000 fold higher than that on healthy epithelial cells. Due to the lower expression level of HER2 on healthy cells and peptides' short dissociation half-life, TNP^HER2pep presenting a moderate affinity targeting peptide, although it may form monovalent interactions, does not have sufficient avidity to stay attached to a healthy cell surface for a time duration that would accommodate endocytosis. Conversely, the same particle is able to simultaneously form multiple interactions preferentially with a breast cancer cell with overexpressed HER2, resulting in avidity enhancement and HER2-mediated endocytosis. (B) To enhance accumulation and cellular uptake of the TNP^HER2pep at the tumor site, it is necessary to optimize the nanoparticle design parameters, such as peptide density and EG linker length, using both in vitro and in vivo methods.

For optimized TNP^HER2pep, we first designed a HER2pep-lipid conjugate since it is the main component of TNP^HER2pep that significantly affects efficient cellular binding and uptake (Fig. 2A). HER2pep was conjugated to three lysines (K3) with an EG2 linker. The EG2 linker functions as a spacer that minimizes the interaction between the oligolysines and the peptide. Lysine residues enhance the hydrophilicity of targeting peptides, enabling peptides exposure above the polyethylene glycol (PEG) coating, thereby improving the peptide availability for HER2 binding. The HER2pep-EG2 spacer-K3 moiety was then connected to palmitic acid lipids via an EG linker to generate HER2pep-lipid conjugate (HER2pep-K3-[EG_n]-lipid, where n is the repeating unit of EG and is either 8 or 18; Fig. 2A). Palmitic acid was chosen for the hydrophobic lipid tails due to its high chemical stability from the lack of phosphoester bonds and its improved solubility in reagents used in solid phase synthesis [28,29]. For the EG linker, we compared EG18 and EG8 linkers. The EG18 linker previously improved peptide flexibility for more efficient binding to the target receptor [30]. We additionally chose the EG8 linker for in vitro and in vivo evaluations, since the EG8 linker forms its structure in a more linear manner than EG18, enhancing peptide accessibility for the target receptor.

Fig. 2.

Design, preparation, and characterization of Dox prodrug-loaded TNP^HER2pep. (A) Structure of HER2pep-K3-[EG_n]-lipid. HER2pep was first conjugated to three lysines (K3) with an EG2 spacer. Then, the HER2pep-EG2-K3 was connected to palmitic acid lipid tails via EG_n linker, where n is the repeating unit of ethylene glycol (n = 8 or 18). (B) Synthetic scheme for Dox-lipid conjugate. Dox-lipid was synthesized by conjugating Dox to DPPE-GA via the acid-labile hydrazone bond. (C) Formulation of TNP^HER2pep. The Dox-lipid, HER2pep-K3-[EG₈]-lipid (or HER2pep-K3-[EG₁₈]-lipid), DSPC bulk lipid, mPEG2000-DSPE lipid conjugate, and cholesterol were mixed at precise stoichiometric ratios, completely dried to form a thin lipid film, rehydrated in PBS, and extruded through a polycarbonate membrane to form the Dox prodrug-loaded TNP^HER2pep. Note that TNP^HER2pep formulated with HER2pep-K3-[EG₈]-lipid and HER2pep-K3-[EG₁₈]-lipid were named as TNP^HER2pep[EG₈] and TNP^HER2pep[EG₁₈], respectively. (D) Dynamic light scattering analysis of nanoparticles showed that all the nanoparticle formulations had an average diameter of ~80 nm. (E) and (F) Peptide and drug loading efficiency. The actual concentrations of HER2pep (blue (E)) and Dox (navy (F)) in the 0.5% TNP^HER2pep[EG₈] were measured and compared with their respective intended loading concentrations of HER2pep (green (E)) and Dox (orange (F)), demonstrating over 98% efficiency in both peptide and drug loading. Data shown are from a representative experiment. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

HER2pep-K3-([EG₁₈]/[EG₈])-lipids were synthesized using Fmoc chemistry-based solid phase peptide synthesis method (Fig. S1). While palmitic acids generated hydrophobic lipid tails that would embed the molecule into the lipid bilayer of the liposomes, HER2pep would be exposed beyond the PEG coating for binding to its target receptor, HER2. The HER2pep-K3-[EG₁₈]/[EG₈]-lipid molecules were purified via RP-HPLC. In parallel, Dox prodrug-lipid (Dox-lipid) was synthesized by the conjugation of Dox to a polar head group of DPPE-GA lipid via an acid-labile hydrazone bond (Fig. 2B), followed by purification by chloroform extraction. The HER2pep-K3-[EG₁₈]/[EG₈]-lipids and Dox-lipid were then characterized by mass spectrometry, and their purities were determined to be > 95% by analytical HPLC with the C3 semi-preparative column (Fig. S2, S3, and Table S1). The hydrazone bond used in conjugation of Dox to lipids helps prevent premature release of Dox in blood circulation (pH = 7.4) and, therefore, prevents non-specific toxicity, since release of active Dox preferentially takes place in an acidic environment (pH = 5.5) of endosomes upon endocytosis of nanoparticles by tumor cells [26,32].

TNP^HER2pep was prepared using a thin lipid film method as shown in Fig. 2C. Briefly, HER2pep-K3-[EG₁₈] (or [EG₈])-lipid was incorporated into liposomal platforms with other lipid components at the following molar ratios: (95-x-y)%:5%:x%:y% of DSPC/mPEG2000-DSPE/DiD/HER2pep-K3-[EG₁₈] (or [EG₈])-lipids, respectively, where x and y represent mole percent (mol%) of DiD fluorescent dye (0.1 ≤ x ≤ 0.75) and mol% of HER2pep density (0.1 ≤ y ≤ 4), respectively. DSPC plays a role in bulk lipid of liposomes. mPEG2000 generates a globular type conformation on the nanoparticle surface, also known as PEG cloud, to increase in vivo half-life by preventing RES clearance, while DSPE lipid is anchored in a lipid bilayer. TNPs incorporated with HER2pep-K3-[EG₈]-lipid and HER2pep-K3-[EG₁₈]-lipid were named as TNP^HER2pep[EG₈] and TNP^HER2pep[EG₁₈], respectively. For Dox prodrug-loaded TNP^HER2pep, we incorporated 2 mol% of Dox-lipid into the liposomes instead of the DiD dye. The cholesterol component was always 5 mol% of total phospholipid concentration to achieve an increased liposomal stability.

TNP^HER2pep was then extruded using a polycarbonate membrane to generate unilamellar liposomes and characterized by dynamic light scattering (DLS) analysis. The DLS results showed that our TNP formulation method yielded nanoparticles with an average diameter of ~80 nm with narrow polydispersity (Fig. 2D), demonstrating that our approach generated homogenous particle populations with minimized batch-to-batch inconsistency. The ~80 nm size was chosen in our study since nanoparticles with smaller size have shown an enhanced tumor penetration as compared to those with larger size [33-36]. We also prepared nanoparticles without targeting peptides (NP) as controls for in vitro cellular uptake studies and in vivo particle tumor cellular uptake and tumor accumulation studies. Furthermore, we prepared Dox prodrug-loaded nanoparticles without HER2pep (NP_Dox) as a control for the in vivo efficacy study.

To confirm that the Dox prodrug-loaded TNP^HER2pep maintained the precise molar ratio of peptide and Dox on each nanoparticle, we investigated loading efficiency via RP-HPLC analysis. We formulated nanoparticles with 2% Dox and 0.5% peptide and compared the concentration of peptide and Dox with the respective theoretical maximum concentration. The Dox prodrug-loaded TNP^HER2pep had a loading efficiency of > 98% for both peptide and drug, suggesting that this method preserves the precise number of peptide and drug molecules per particle during nanoparticle formulation (Fig. 2E, F). Collectively, our synthetic approach generated highly homogeneous particles that had high peptide and drug loading efficiency as well as narrow size range precision with minimized batch-to-batch variability.

2.2. In vitro evaluation of cellular uptake of TNP^HER2pep by HER2-positive human and neu-positive mouse breast cancer cells

Peptide density and EG linker length are the two most impactful design parameters of peptide-targeted nanoparticles that influence the efficiency of target binding and cellular uptake [28-30]. First, to evaluate the effect of peptide density on cellular uptake efficiency, we prepared TNP^HER2pep[EG₁₈] at varying peptide densities (0.1–4%). Cells treated with non-targeted nanoparticles (NP; 0% peptide density) and untreated cells (UT) were included as additional controls. 0.1 mol% DiD fluorescent dye was incorporated into all nanoparticles for quantification of nanoparticle cellular uptake by flow cytometry. HER2 overexpressing BT-474 and SK-BR-3 breast cancer cell lines were incubated with different nanoparticle formulations for 3 h, and uptake was analyzed by flow cytometry. The MCF-7 cell line, which expresses very low levels of HER2, was used as a negative control.

In all human and mouse breast cancer cell lines tested, there was no detectable fluorescence difference between NP and UT, demonstrating that NP was not taken up by the cells, DiD containing particles were stable, and DiD dye was unlikely to be released from the particles (Fig. 3A and B). Our results showed that the cellular uptake of TNP^HER2pep did not significantly change at 0.1–0.7% peptide density in all human breast cancer cells (Fig. 3A). However, as peptide density increased to and above 1%, the cellular uptake of TNP^HER2pep increased significantly for both HER2+ BT-474 and SK-BR-3 cell lines (Fig. 3A). At 1% peptide density, the cellular uptake of TNP^HER2pep was increased ~11 fold and ~20 fold over NP in BT-474 and SK-BR-3 cell lines, respectively, while the cellular uptake observed in the MCF-7 cell line was negligible (Fig. 3A). The maximum cellular uptake was at 2% peptide density and then plateaued, presumably due to particle saturation on the cellular surface (Fig. 3A). These results indicated that a 1–2% peptide density is the optimal peptide density for selective and efficient cellular uptake in vitro.

Fig. 3.

In vitro evaluation of cellular uptake of TNP^HER2pep by HER2-positive human and neu-positive mouse breast cancer cells. (A) The effect of peptide density on uptake of TNP^HER2pep by HER2-positive human breast cancer cell lines. In vitro cellular uptake of TNP^HER2pep[EG₁₈] formulations of various peptide densities (0.1%–4%) was tested using HER2 positive human breast cancer cell lines: BT-474 and SK-BR-3. MCF-7, which is a HER2 negative human breast cancer cell line, was used as a negative control. Cells treated with non-targeted nanoparticles (NP) and untreated cells (UT) were also used as additional controls. All experiments were repeated in triplicate and data represents means ( ± s.d.). In all cell lines, fluorescence observed in NP was not statistically different from UT (N.S.; not significant). Asterisk (*) and pound (^#) represent statistical significance of cellular uptake by SK-BR-3 and BT-474 at 1% peptide density, respectively, as compared to 0.7% peptide density and NP (p < .001). (B) The effect of EG linker length on uptake of TNP^HER2pep by HER2/neu-positive mouse breast cancer cell line. In vitro cellular uptake of TNP^HER2pep[EG₈] and TNP^HER2pep[EG₁₈] with varying peptide densities (0.7%–4%) was evaluated using neu-expressing mouse mammary tumor epithelial cells. Cells treated with non-targeted nanoparticles (NP) and cells without treatment of nanoparticles (UT) were also used as controls. In all experiments, near infrared DiD dye (0.1 mol%) was loaded in TNP^HER2pep to aid in nanoparticle uptake quantification. Fluorescence signal (RFU) was analyzed by flow cytometry. All experiments were done in triplicate, and data represents means ( ± s.d.). No statistical difference of fluorescence was observed in groups between UT and NP (N.S.; not significant). Asterisk (*) indicates statistical significance of cellular uptake of TNP^HER2pep[EG₈] at 1% peptide density in comparison with 0.7% peptide density and NP (p < .001), while pound (^#) represents statistical significance of cellular uptake of TNP^HER2pep[EG₁₈] at 2% peptide density as compared to lower peptide densities (0.7% and 1%) and NP (p < .001). The P values were calculated using a student's t-test.

To investigate whether TNP^HER2pep exerts cytotoxic effect selectively on HER2+ cells over HER2− cells upon cellular uptake, we used an in vitro cytotoxicity experiment by treating SK-BR-3 (HER2+) and MCF-7 (HER2−) cell lines with a Dox-prodrug loaded TNP^HER2pep. The results showed that the Dox-prodrug loaded TNP^HER2pep effectively killed HER2+ cells with an IC₅₀ of ~2.5 μM, whereas it was not significantly cytotoxic to HER2− cells, even at 10 μM Dox equivalent concentration, demonstrating selective and effective cytotoxic effect of TNP^HER2pep on HER2+ cells (Fig. S4).

Next, we evaluated the effect of EG linker length on cellular uptake efficiency in vitro. For this purpose, we used primary Her2/neu-positive (neu+) mouse mammary tumor epithelial cells that were isolated from a tumor tissue generated by the MMTV-neu transgenic mouse. This cell line provided two advantages. First, the cell line closely mimics HER2+ breast cancer cells due to the high sequence homology between mouse neu/Her2 to human HER2 [37-39]. Second, the cell line provides an in vitro evaluation tool related to the in vivo assessment, since the cells were directly derived from the MMTV-neu spontaneous tumor, which were used for the in vivo animal study.

Although neu is highly homologous to HER2, we validated that TNP^HER2pep was effectively taken up by the neu cell line (Fig. 3B). To evaluate the effect of EG linker length on cellular uptake, we used an in vitro cellular uptake assay with the neu-positive breast tumor cells. We prepared TNP^HER2pep[EG₈] and TNP^HER2pep[EG₁₈] at 0.7–4% peptide density. Non-targeted nanoparticles (NP) were also used as a control. All the nanoparticles were loaded with 0.1 mol% DiD fluorescent dye for quantification of nanoparticle cellular uptake by flow cytometry.

Together, these results demonstrated that TNP^HER2pep was effectively taken up by neu+ mouse mammary cancer cells, presumably due to neu's more than 90% primary sequence homology to HER2 (Fig. 3B) [37-39]. Importantly, the results showed that TNP^HER2pep[EG₈] yielded significant enhancement of cellular uptake with ~15 fold to ~76 fold higher levels relative to NP, as peptide density increased from 1% to 4% (Fig. 3B). Interestingly, even at high peptide densities (2–4%), the TNP^HER2pep[EG₁₈] exhibited relatively less enhancement in cellular uptake, reaching ~10 fold higher levels over NP (Fig. 3B). This could be presumably due to longer linker's less linear type conformation, which might increase the fraction of peptides hindered within PEG cloud. Overall, the in vitro results from both HER2+ human and neu+ mouse breast cancer cellular uptake studies demonstrated that the EG8 linker and 1–2% peptide densities provide the optimal parameters that result in efficient cellular uptake of TNP^HER2pep in vitro.

2.3. Effect of peptide density and EG linker length on in vivo tumor accumulation and tumor cell uptake of TNP^HER2pep

In vitro studies provide insight to optimization of nanoparticle design parameters. Nevertheless, without further in vivo evaluation, in vitro optimization is insufficient to dissect and identify the exact nanoparticle parameters that will yield optimum patient outcomes. Although our in vitro results suggested that EG8 and 1–2% peptide densities are the optimal EG linker length and peptide densities for selective and efficient cellular uptake in vitro, respectively, this may not directly correlate with in vivo results, since shorter linker length and increased peptide density may clear the TNP^HER2pep from blood circulation by RES. Hence, nanoparticle formulation parameters still needed to be further optimized in vivo. Therefore, we next tested our in vitro cellular uptake results in vivo and further optimized the TNP^HER2pep by investigating the two endpoints: i) in vivo nanoparticle accumulation at tumor site and ii) in vivo cellular uptake by tumor cells following accumulation at tumor tissues.

Whole-body imaging studies help compare the relative delivery of different nanoparticles in each organ. Nevertheless, please note that the fluorescence intensity does not necessarily show a linear relationship with the dye level in each tissue; thus, the whole-body imaging is at most meaningful as a semi-quantitative measure and should not be considered a conclusive biodistribution study. A more appropriately performed biodistribution study would involve a validated calibration curve of the signal in each tissue vs. concentration of the analyte. We have previously validated that fluorescence measurements from dissected organs by the imager exactly matched the calibrated fluorescence readings obtained via extraction of the dye into organic solvents from homogenized organs. Therefore, here, we compared normalized fluorescence measurements by each organ's weight to obtain a more accurate and directly comparable nanoparticle tissue biodistribution.

We examined nanoparticle tissue biodistribution and tumor cellular uptake using an in vivo MMTV-neu transplantation model (Fig. S5A) and the TNPs with varied density and linker length. These samples included non-targeted nanoparticles (NP) and TNP^HER2pep with various peptide densities (0.25%, 0.5%, 0.7%, and 1%) and either EG8 or EG18 linker (0.25% TNP^HER2pep[EG₈], 0.5% TNP^HER2pep[EG₈], 0.7% TNP^HER2pep[EG₈], 1% TNP^HER2pep[EG₈], 0.25% TNP^HER2pep[EG₁₈], 0.5% TNP^HER2pep[EG₁₈], 0.7% TNP^HER2pep[EG₁₈], and 1% TNP^HER2pep[EG₁₈]). All nanoparticles were loaded with DiD dye for fluorescence quantification of both nanoparticle tumor accumulation and cellular uptake.

For nanoparticle accumulation in the tumors, we detected fluorescence of tumor tissue using in vivo IVIS Lumina II imager and then analyzed TNP tumor accumulation using Image J. For tumor cell uptake studies, we disaggregated tumor tissues into a single cell suspension and analyzed relative fluorescence by flow cytometry. TNP^HER2pep with higher than 1% peptide density were not evaluated in vivo, since our former studies demonstrated that higher peptide densities have non-selective and off-target interactions to healthy cells in vivo [31].

Our TNP tumor accumulation results showed that at low peptide densities (0.25–0.5%), TNP^HER2pep[EG₈] accumulated in tumor tissue with a comparable amount to control NP (Fig. 4A, left). As the peptide density increased to 0.7–1%, the TNP tumor accumulation decreased (Fig. 4A, left). The cohorts treated with TNP^HER2pep[EG₁₈] had a similar trend in TNP tumor accumulation (Fig. 4A, right). At 0.25–0.5% peptide density, TNP^HER2pep[EG₁₈] showed similar levels of TNP tumor accumulation, while TNP tumor accumulation decreased compared to the control group treated with NP, as the peptide density increased to 0.7–1% (Fig. 4A, right). These results demonstrated that peptide densities above 0.5% prevented TNP^HER2pep from preferentially localizing at tumor site, presumably by off-target interactions and RES clearance.

Fig. 4.

Effect of peptide density and EG linker length on in vivo tumor accumulation and tumor cell uptake of TNP^HER2pep. Tumor-bearing NOD-SCID mice were intravenously injected with TNP^HER2pep[EG₈] and TNP^HER2pep[EG₁₈] of various peptide densities (0.25%, 0.5%, 0.7%, and 1%). NP (0% peptide density) was used as a negative control. DiD dye was incorporated in each nanoparticle for quantification of tumor accumulation and tumor cell uptake. All data shown are means ( ± s.d.) of n = 5 per treatment group. (A) Tumor accumulation of TNP^HER2pep. 24 h after nanoparticle injections, mice were sacrificed, tumor tissues were dissected, and nanoparticle accumulation was analyzed by in vivo IVIS Lumina II imager and Image J. Results of TNP^HER2pep engineered with EG8 (left panel) and EG18 (right panel) linkers. Asterisk (*) indicates the peptide density at which in vivo TNP^HER2pep tumor accumulation was significantly reduced as compared to NP (p < .001). (B) Tumor cell uptake of TNP^HER2pep. 24 h after nanoparticle treatment, mice were sacrificed, tumor tissues were dissected and disaggregated to a single cell using tumor dissociation solution comprising collagenase and DNase. Tumor cell uptake of TNP^HER2pep was then analyzed using flow cytometry. Left panel shows tumor cell uptake of TNP^HER2pep[EG₈], and right panel shows tumor cell uptake of TNP^HER2pep[EG₁₈] at 0.25–1% peptide density. Pound (^#) and asterisk (*) represent statistical significance of tumor cell uptake at 0.25% and 0.5% peptide density, respectively, as compared to NP (p < .001). A student's t-test was used for P values.

In our organ biodistribution study, although we did not observe a dramatic increase in accumulation at the spleen in various animal groups with increased peptide density, this might have been due to animals being sacrificed 24 h post-injection for biodistribution analysis, and TNPs with high peptide densities were potentially already mostly collected by the spleen and cleared by excretion (Fig. S6). Nevertheless, the results demonstrated a clear trend of increased fluorescence in spleen with all groups treated with TNP^HER2pep, compared to NP group, suggesting clearance of TNP^HER2pep mostly by spleen (Fig. S6). Importantly, the results showed no increased fluorescence in other organs from all TNP^HER2pep groups relative to NP group, demonstrating a lack of off-target accumulation of TNP^HER2pep in any organs (Fig. S6).

We next evaluated the in vivo cellular uptake at the tumor site to see if TNP^HER2pep provided any advantages for efficient intracellular delivery of the therapeutic agents. Our results demonstrated a dramatic enhancement of uptake with TNP^HER2pep[EG₈] over NP group, specifically ~2.3 fold and ~2.7 fold with 0.25% and 0.5% peptide density, respectively (Fig. 4B, left). Similarly, at 0.25–0.5% peptide density, TNP^HER2pep[EG₁₈] showed a significant enhancement of uptake, reaching to ~1.7 fold and ~3.4 fold higher levels over NP, respectively (Fig. 4B, right). Taken together, these results demonstrated that while TNP^HER2pep showed comparable tumor accumulation to NP at 0.25–0.5% peptide density, the cellular uptake at the tumor site was significantly enhanced with both TNP^HER2pep[EG₈] and TNP^HER2pep[EG₁₈] at 0.25–0.5% density. This suggests that for TNP^HER2pep, 0.25% and 0.5% peptide density with either EG8 or EG18 linker provide the optimal design parameters that could enhance localization and intracellular uptake of therapeutics for improved patient outcomes.

2.4. Effect of peptide density and EG linker length on in vivo efficacy and toxicity of Dox prodrug-loaded TNP^HER2pep

Based on the biodistribution results, tumor accumulation, and tumor cell uptake studies, we observed that both tumor accumulation and tumor cell uptake were significantly reduced in groups treated using TNP with higher peptide density (> 0.5%). Given this result, we concluded that TNP formulations with over 0.5% peptide loading would not achieve enhanced efficacy. Meanwhile, during the tumor cell uptake study, we observed significantly enhanced uptake for TNP formulations with 0.25% to 0.5% peptide density. Although the result suggested that tumor cell uptake reached a maximum with 0.5% peptide loading, we investigated the possibility of a formulation with a peptide density between 0.25% and 0.5% that could have achieved higher tumor cell uptake, which presumably would accomplish enhanced in vivo efficacy. Therefore, we additionally formulated a 0.35% peptide loaded TNP group for further in vivo efficacy studies to examine whether this formulation would improve in vivo outcomes.

To evaluate the efficacy of tumor inhibition by the optimized TNP^HER2pep in vivo, we used an orthotopic MMTV-neu transplantation animal study (Fig. S5B). For this study, 2 mol% Dox-lipid was incorporated into each TNP^HER2pep, and mice were distributed into 9 treatment groups: PBS (Control), free Dox (Dox), non-targeted Dox prodrug-loaded nanoparticles (NP_Dox), and Dox prodrug-loaded TNP^HER2pep with variable peptide densities (0.2%, 0.35%, and 0.5%) and either EG8 or EG18 linker (0.2% TNP^HER2pep[EG₈]_Dox, 0.35% TNP^HER2pep[EG₈]_Dox, 0.5% TNP^HER2pep[EG₈]_Dox, 0.2% TNP^HER2pep[EG₁₈]_Dox, 0.35% TNP^HER2pep[EG₁₈]_Dox, and 0.5% TNP^HER2pep[EG₁₈]_Dox).

Our in vivo efficacy results showed that while NP_Dox treatment reduced the tumor burden, the tumor burden continued to increase over time, suggesting its limited efficacy in reducing, but not completely inhibiting, tumor growth (Fig. 5A). Importantly, TNP^HER2pep[EG₈]_Dox at all of the tested peptide densities dramatically inhibited tumor growth, compared with PBS control and NP_Dox (Fig. 5A, left), while the TNP^HER2pep[EG₈]_Dox and free Dox had similar efficacy. Interestingly, TNP^HER2pep[EG₁₈]_Dox (Fig. 5A, right) also dramatically inhibited tumor growth with 0.35% and 0.5% but was less inhibitory at the 0.2% peptide density, suggesting that the EG18 linker was less effective at inhibiting tumor growth than was the EG8 linker. This difference in efficacy based on the linker length is likely due to EG8 linker's more linear conformation that enables a greater fraction of peptides to be presented above PEG cloud, making it more available for binding to the target receptor as compared to EG18 linker, despite peptide density being the same for both linkers on the nanoparticle surface. Overall, these results demonstrated that TNP^HER2pep[EG₈]_Dox at 0.2–0.5% peptide density and TNP^HER2pep[EG₁₈]_Dox at 0.35–0.5% peptide density dramatically reduced tumor growth relative to controls, presumably due to improved tumor cell uptake with optimized peptide density and linker length.

Fig. 5.

Effect of peptide density and EG linker length on in vivo efficacy and toxicity of Dox prodrug-loaded TNP^HER2pep. Tumor-bearing NOD-SCID mice were intravenously injected on days 1, 3, 5, 7, and 9 with Dox prodrug-loaded TNP^HER2pep[EG₈] and TNP^HER2pep[EG₁₈] with various peptide densities (0.2%, 0.35%, and 0.5%). For this, 2 mol% Dox-lipid was incorporated into all nanoparticles, and a Dox equivalent of 3 mg/kg was injected for each treatment. Free doxorubicin (Dox), NP_Dox (non-targeted), and PBS were used as controls. Tumor volume was calculated via tumor measurements with a caliper. All data shown are means ( ± s.d.) of n = 7 per treatment group. (A) The effect of peptide density and linker length on antitumor efficacy of TNP^HER2pep. (Left) Tumor growth suppression by TNP^HER2pep[EG₈], and (Right) Tumor growth suppression by TNP^HER2pep[EG₁₈]. The arrows indicate treatment injection days. On day 24, tumor volumes of all mice groups treated with Dox prodrug-loaded TNP^HER2pep[EG₈] formulations of different peptide loadings were not statistically different from each other, while the tumor volumes in these groups were statistically significant compared to NP_Dox (asterisk (*); p < .001). For the mice treated with the longer linker formulation (Dox prodrug-loaded TNP^HER2pep[EG₁₈]), the tumor volumes were not statistically different for 0.35% and 0.5% peptide loading formulations. However, tumor volume differed significantly for 0.2% peptide loaded formulation, which was closer to NP_Dox treated group (asterisk; p < .001). A student's t-test was used for statistical comparison. (B) Systemic toxicity evaluation via change in percent body weight. Change in body weight (%) of the same animal groups for the efficacy study are indicative of systemic toxicity during treatment with TNP^HER2pep[EG₈] (left) and TNP^HER2pep[EG₁₈] (right) at 0.2, 0.35 and 0.5% peptide densities. All mice in the free Dox group had to be sacrificed on day 9, due to significant systemic toxicity (> 15% loss in body weight), while body weight of the other treatments group remained unchanged during the study. Pound (^#) indicates no statistical difference for change in body weight of mice for all nanoparticle treatment groups (including both EG8 and EG18 linkers) as well as the control group. A student's t-test was used for P values.

To evaluate systemic toxicity in vivo, mouse body weight was measured throughout the study (Fig. 5B). The free Dox group showed significant toxicity during the study and was sacrificed on day 9, due to a significant weight loss (> 15%). The other groups saw no detectable change of body weight. Additionally, only Dox treatment induced a significant reduction in organ weight (specifically kidneys, spleen, and liver), while all the TNP treatment cohorts had no-to-minor impact on organ weight (Fig. S7). Overall, these results demonstrate that TNP^HER2pep[EG₈]_Dox and TNP^HER2pep[EG₁₈]_Dox significantly reduced systemic toxicity compared to free Dox treatment.

Taken together, our results demonstrated both efficacy at reducing tumor burden and reduced toxicity in vivo. Dox prodrug-loaded TNP^HER2pep dramatically enhanced therapeutic efficacy over NP_Dox via optimization of the crucial nanoparticle design parameters, peptide density and linker length. Significantly, these TNPs also reduced overall systemic toxicity over free Dox by preventing premature drug release into circulation due to their tumor cell-selective intracellular drug delivery. These results suggest that optimally designed TNP^HER2pep will have efficacy that surpasses both conventional chemotherapeutics and non-targeted nanoparticles.

3. Discussion

For over a decade, HER2 TNP drug delivery has improved therapeutic efficacy across many different preclinical studies, however, no examples have successfully translated into the clinic [4-13]. This is primarily due to two significant hurdles: i) the use of high affinity targeting moieties, such as anti-HER2 antibodies, resulting in reduced selectivity for HER2+ tumor cells over healthy cells; and ii) batch-to-batch inconsistency in nanoparticle production resulting in significant variations in clinical outcomes and limiting scalability for commercialization.

To improve the selectivity of HER2 TNPs, we utilized a cyclic peptide (HER2pep) with weak-to-moderate affinity and used a multivalent strategy to gain avidity only for those cells with overexpressed HER2 receptors. To successfully accomplish this strategy, it is critical to have a precise control over the valency of the HER2pep on the particle surfaces. Hence, we utilized a synthetic strategy that reproducibly generated highly pure and precisely controlled Dox prodrug-loaded TNP^HER2pep formulations. This was accomplished by pre-synthesizing the peptide-lipid and drug-lipid conjugates and by subsequently incorporating their purified forms into a liposomal platform with pure lipid constituents at specific molar ratios prior to hydration. In this study, after employing this synthetic strategy, we systematically analyzed the effects of individual nanoparticle design parameters, specifically peptide density and peptide linker length, on in vitro and in vivo outcomes to identify the optimal TNP^HER2pep formulations with enhanced selectivity for enhanced antitumor efficacy. This synthesis approach is expected to generate significantly more consistent in vitro and in vivo experimental outcomes, resulting in more reliable preclinical rationales for clinical studies.

Although in vitro evaluation provides a good starting point for therapeutic efficacy, in vitro results do not directly translate into in vivo outcomes, due to the increased complexity under in vivo circumstances, including a blood flow affecting interactions between peptide and target receptor, off-target binding to healthy cells, RES clearance, nanoparticle tumor accumulation via enhanced permeability and retention (EPR) effect, tumor penetration, and binding site barrier. As the first step in optimizing design parameters, in vitro evaluations are necessary prior to in vivo studies to predict trends of how the parameters would affect in vivo outcomes to eliminate potential negative consequences. Our in vitro studies, unsurprisingly, revealed that increasing peptide density results in more efficient binding and uptake of nanoparticles. We determined a threshold peptide density of 1–2%, which leads to nonselective cellular binding/uptake by HER2-negative cells (MCF-7), given a clear indication of non-selective binding/uptake at 2% peptide density. Further in vitro analysis using varied peptide linker lengths demonstrated the dramatically increased cellular uptake by shorter linker length (EG8) but undetectable cellular uptake by longer linker length (EG18) at 1% peptide density. The efficient display of the peptides, thus, prevented their obstruction by PEG or lipid and enhanced their receptor binding.

Our in vivo nanoparticle tumor accumulation study directly revealed the necessity of in vivo optimization following in vitro studies. While ~1% peptide density was considered the optimal peptide density by in vitro results, this density significantly reduced the particle accumulation in vivo in the tumors for both TNP^HER2pep[EG₈] and TNP^HER2pep[EG₁₈]. Rather, lower peptide densities at 0.25–0.5% enabled TNPs to take advantage of EPR effect more efficiently for preferential tumor accumulation, suggesting that higher peptide densities result in increased probability of off-target binding to healthy cells and RES clearance. This was supported by a published study reporting that decreased particle accumulation in tumors and increased clearance by RES organs were associated with high surface targeting element density [40]. Most important, further analysis of in vivo nanoparticle tumor cell uptake demonstrated that at 0.25–0.5% peptide densities, the optimally designed TNP^HER2pep[EG₈] and TNP^HER2pep[EG₁₈] are effectively taken up, even while preserving their ability to accumulate at tumor via EPR effect. Given the mice used for this study were the NOD-SCID model (NOD: defective innate immune cells; SCID: lacking in B & T cells), enhanced tumor cell uptake in optimal TNP formulations was achieved primarily due to HER2 receptor activated endocytosis [41]. Furthermore, the results established that, with optimally designed TNP^HER2pep, a larger amount of drug is delivered inside the tumor cells, relative to non-targeted nanoparticles, even if the total amount of drug accumulation at the tumor is similar.

The direct comparison of linker length with tumor cell uptake indicated a complex relationship between linker length and peptide density in promoting targeted cellular uptake. The in vivo cellular uptake of TNPs was similar at both 0.25% and 0.5% peptide densities when using the EG8 linker, whereas the cellular uptake of TNPs was much higher at 0.5% peptide density than at 0.25% when using the EG18 linker (Fig. 4B). We hypothesize that the EG8 linker forms a more linear, rigid conformation that increased the fraction of peptides exposed, making them available continually for receptor binding, presumably resulting in TNPs-HER2 receptor binding reaching saturation even at lower peptide density (0.25%). In contrast, the EG18 linker forms a relatively less linear and less rigid conformation that increases the fraction of peptides buried within the PEG cloud, thus, achieving particle-receptor binding saturation at a higher peptide density (0.5%). Interestingly, in vivo the cellular uptake decreased significantly at 0.5–0.7% peptide density, regardless of linker lengths, despite a similar tumor accumulation. This was likely due to the binding site barrier phenomenon, in which nanoparticles with higher peptide densities are likely to be trapped within the first layer of the cells located close to angiogenic vasculature, due to their excessively high affinities and unintended binding to these cells and proteins, which decreases the number of particles that penetrate into the tumor tissue [42]. This result implies an upper limit of peptide density for particles to penetrate deep into interstitial space, which is required to effectively kill the tumor cells.

Even though targeted nanoparticle delivery has been viewed as a game changer, few nanoparticle delivery systems have made it past Phase I clinical trials, and none have passed Phase II [9]. Unfortunately, only a very small percentage of the nanoparticles ends up actually endocytosed by the tumor cells. A recent report found as little as 0.0014% of the TNP made it into the tumor cells, with a very low level of accumulation (0.7%) at tumor tissue [8]. The accumulation and uptake by unintended cells may be due to flaws in particle design and production approaches that use high affinity targeting moieties, which hinder TNPs from selectively accumulating and being taken up by cancer cells. Consistent with the current results, our previous study also demonstrated that TNPs with high avidity significantly reduced both tumor accumulation and tumor cell uptake [31]. In the current study, we have evaluated the contributions of important nanoparticle design parameters, both in vitro and in vivo, to create optimal in vivo therapeutic outcomes. Our in vivo antitumor efficacy studies established the superiority of in vivo optimized TNP^HER2pep that is loaded with Dox prodrug compared to non-targeted NP_Dox, which we attribute to the enhanced selectivity in HER2+ tumor cell uptake of the targeted drug delivery. Unsurprisingly, both nanoparticle versions significantly minimize the systemic toxicity compared to free Dox. We predict that our optimized drug delivery system will outperform other TNP drug delivery systems that use high affinity targeting moieties. In the future, we expect targeted nanoparticle optimization, as described in this paper, to become a standard method of developing targeted nanoparticle formulations with improved selectivity and reproducibility.

While HER2 targeted therapies are particularly more effective in combination with chemotherapy, the combination therapy is not always a clinical option for HER2+ breast cancer patients. Specifically, for those patients who have underlying health issues, such as heart disease, significant toxicities from some of the chemotherapies and therapeutic agents may increase mortality, rather than improving outcome. We believe our tumor cell selective targeting approach with TNP^HER2pep will enable the use of combination therapy in a broader patient population due to the low systemic toxicity and enhanced efficacy that is available for a larger patient population. Future research will compare our described TNP^HER2pep drug delivery system to the standard-of-care treatment—both HER2 targeting mAb therapy as a single agent and in combination with chemotherapy.

4. Conclusions

Here, we described a two-step approach of TNP^HER2pep optimization to achieve enhanced in vivo tumor inhibition efficacy with minimized toxicity. The results established that the optimized Dox prodrug-loaded TNP^HER2pep formulation outperformed both NP_Dox and free Dox versions by accomplishing enhanced in vivo anti-tumor efficacy with minimal toxicity. Nevertheless, despite a clear evidence of suppression in tumor growth, optimal TNP formulations did not accomplish complete shrinkage of tumor. We predict, however, that given the high therapeutic index in all NP treated groups, the mice receiving treatment with TNP formulations could have tolerated much higher drug concentrations than administered. In which case, the efficacy of Dox prodrug-loaded TNP^HER2pep would have further improved possibly to complete eradication of the HER2+ breast tumor. Furthermore, we expect TNP^HER2pep to perform significantly better compared to anti-HER2 antibody TNP drug delivery systems because of the superiority in precision and reproducibility in targeted-nanoparticle synthesis, and deliver consistent experimental outcomes. In conclusion, this study highlights the significance of systematic optimization of nanoparticle design parameters for tumor selective drug delivery and indicates that this approach can achieve a high therapeutic potential of Dox prodrug-loaded TNP^HER2pep for HER2+ breast cancer in the clinic.

5. Methods

5.1. Materials

We purchased NovaPEG Rink Amide Low Loading Resin, 2-(1H-benzotriazol-1-yl)-1,1,3,3 tetramethyluroniumhexafluorophosphate (HBTU), and all Fmoc-protected amino acids from EMD Millipore. We purchased N,N-diisopropylethylamine (DIEA), trifluoroacetic acid (TFA), triisopropylsilane (TIS), dimethylformamide (DMF), dichloromethane (DCM), 2-proponol (IPA), acetonitrile (AcN), ethanol, Kaiser test reagents, cholesterol, N,N′-diisopropylcarbodiimide (DIC), hydrazine, chloroform, doxorubicin hydrochloride, and DNase I from Sigma-Aldrich. 1,2-ethanedithiol (EDT) was purchased from Alfa Aesar. We obtained 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(glutaryl) (DPPE-GA), 1,2-distearoyl-sn-glycero-3-phosphocholine (sodium salt) (DSPC), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (mPEG2000-DSPE) from Avanti Polar Lipids. 3H-indolium, 2-(5-(1,3-dihydro-3,3-dimethyl-1-octadecyl-2H-indol-2-ylidene)-1,3-penta-dienyl)-3,3-dimethyl-1-octadecyl-, perchlorate (DiD fluorescent dye) was acquired from Invitrogen. Fmoc-EG₂-OH, Fmoc-EG₆-OH, and Fmoc-EG₈-OH were purchased from Quanta Biodesign. Collagenase type IV was purchased from Life Technologies.

5.2. Synthesis and characterization of HER2pep-lipid conjugates

HER2pep-lipid (HER2pep-K3-[EG_n]-lipid, where n is the repeating unit of ethylene glycol and is either 8 or 18) conjugates were synthesized by Fmoc chemistry based-solid phase peptide synthesis method as previously described [28-30]. The peptide sequence is YCDGFYACY-MDV. The HER2pep-lipid conjugates were cleaved from resin and purified using a Zorbax C3 semi-preparative column and Agilent 1200 RP-HPLC with a gradient of 60–80% for 10 min in a two-phase system of IPA/AcN/H₂O mixture and H₂O. The products were characterized by Bruker microTof-Q II, and their purities (> 95%) were determined by the RP-HPLC analytical injections using a Zorbax C3 semi-preparative column. Peptide cyclization through disulfide bond formation was conducted with overnight stirring. After cyclization, the products were re-purified using RP-HPLC with the same method used for removal of unreacted products.

5.3. Synthesis and characterization of Dox-lipid conjugates

The Dox prodrug lipid (Dox-lipid) conjugates were synthesized as previously reported [26,32]. Dox-lipids were purified by extraction into chloroform. The products were characterized by Bruker microTof-Q II mass spectrometry, and the purity (> 95%) was determined by RP-HPLC analytical injections through the Zorbax C3 semi-preparative column.

5.4. Preparation of liposomal nanoparticles

The liposomal nanoparticles were prepared via thin film method as previously described [28-30]. Briefly, DSPC, mPEG2000-DSPE, either DiD fluorescent dye or Dox-lipid, HER2pep-K3-[EG₁₈] (or [EG₈])-lipid, and cholesterol were mixed in a glass vial. The solvent was dried using nitrogen gas to form a thin film. The thin film was rehydrated with PBS (pH 7.4) at 70 °C for 10 min and extruded via polycarbonate filter membrane using Avanti Polar Lipid extruder set. The nanoparticles were formulated with the following molar ratio: 1) (94.9-x)%:5%:0.1%:x% of DSPC/mPEG2000-DSPE/DiD/HER2pep-K3-[EG₁₈] (or [EG₈])-lipid, where x is mole percent (mol%) of HER2pep-K3-[EG₁₈] (or [EG₈])-lipid and 0.1 ≤ x ≤ 4, for in vitro cellular uptake assays, 2) (94.25-x)%:5%:0.75%:x% of DSPC/mPEG2000-DSPE/DiD/HER2pep-K3-[EG₁₈] (or [EG₈])-lipid, where x is mol% of HER2pep-K3-[EG₁₈] (or [EG₈])-lipid and 0.25 ≤ x ≤ 1 for in vivo tissue biodistribution and tumor cellular uptake studies, and 3) (93-x)%:5%:2%:x% of DSPC/mPEG2000-DSPE/Dox-lipid/HER2pep-K3-[EG₁₈] (or [EG₈])-lipid, where x is mol% of HER2pep-K3-[EG₁₈] (or [EG₈])-lipid and 0.2 ≤ x ≤ 0.5 for in vivo efficacy studies. The cholesterol used for the entire study was 5 mol% of total phospholipid concentration.

5.5. Liposomal nanoparticle sizing

The size of the nanoparticles was measured using dynamic light scattering (DLS), NanoBrook Omni Particle Size Analyzer (Brookhaven Instruments Corp.), as previously reported [28-30].

5.6. Peptide and drug loading efficiency

Nanoparticles were prepared and purified via liposome extruder purification (LEP) method as previously described [43]. Then, HER2pep-K3-[EG₈]-lipid and Dox-lipid concentration in the TNP^HER2pep were measured via RP-HPLC at 220 nm and 485 nm, respectively. The concentrations were compared with the respective theoretical maximum HER2pep-K3-[EG₈]- lipid and Dox-lipid concentration for peptide and drug loading efficiency, respectively.

5.7. Animals

All animal experiments were conducted with approval by the University of Notre Dame Institution Animal Care and Use Committee guidelines (protocol # 18-11-5000). For transplantation studies, tissue chunks that had been collected from breast tumors generated by MMTV-neu mice were transplanted into the #4 mammary fat pads of NOD.CB17-Prkdc^scid/NCrCrl (NOD SCID; Charles River) recipient mice [44]. Mice used in this study were maintained under pathogen-free conditions in the University of Notre Dame Freimann Life Sciences animal facility.

5.8. Cell culture

BT-474, SK-BR-3, and MCF7 cells were purchased from ATCC (Rockville, MD). Mouse mammary tumor primary epithelial cells used for in vitro studies were isolated from spontaneous tumor bearing MMTV-neu mice [45]. SK-BR-3, MCF7, and BT-474 cells were cultured in RPMI1640 media (Gibco) supplemented with 10% Benchmark fetal bovine serum (Gemini), 2 mM l-glutamine (Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco). Mouse mammary epithelial cells were cultured in DMEM/F-12 (Gibco) supplemented with 10% benchmark fetal bovine serum (Gemini), 5 mL GlutaMAX™ (Gibco), 5 μg/mL Insulin (Sigma), 1 μg/mL Hydrocorticosone (Sigma), 10 ng/mL epidermal growth factor (Gibco), 100 U/mL penicillin (Gibco), and 100 μg/mL streptomycin (Gibco).

5.9. In vitro nanoparticle cellular uptake assay

Nanoparticle cellular uptake assay was described previously [28-30]. 1 × 10⁵ cells/well were plated in a 24-well dish overnight in 37 °C incubator. After overnight incubation, nanoparticles (22.5 μM total phospholipid concentration) were administered to the wells and incubated for 3 h at 37 °C. The 0.1 mol% DiD dye was incorporated into each nanoparticle for quantification of cellular uptake. After 3 h incubation, the cells were washed twice with PBS buffer (pH 7.4) and then trypsinized for 3 min to remove cellular surface-associated nanoparticles. Then, the cells were collected and washed twice with PBS buffer. The cellular fluorescence was analyzed by Guava easyCyte 8HT flow cytometer.

5.10. In vitro cytotoxicity assay

2 × 10⁴ cells/well were plated in a 96-well dish overnight in 37 °C incubator. After overnight incubation, the cells were treated with either free Dox or a Dox-prodrug loaded nanoparticle for 48 h. Cell counting kit-8 (Dojindo) was used for analysis of cell viability. Cells without treatment were used as a control.

5.11. In vivo nanoparticle tissue biodistribution studies

Tumors derived from MMTV-neu transplantation mouse model were used for the in vivo nanoparticle tissue biodistribution study [44]. Spontaneous tumors derived from MMTV-neu transgenic mice were cryopreserved as chunks and later transplanted into #4 mammary glands of NOD-SCID female mice. The mice were randomly distributed into 9 groups (n = 5 per group): Treatment of non-targeted nanoparticles (NP) and HER2 binding peptide presenting targeted nanoparticles with various peptide densities (0.25%, 0.5%, 0.7%, and 1%) and either EG8 or EG18 linker (0.25% TNP^HER2pep[EG₈], 0.5% TNP^HER2pep[EG₈], 0.7% TNP^HER2pep[EG₈], 1% TNP^HER2pep[EG₈], 0.25% TNP^HER2pep[EG₁₈], 0.5% TNP^HER2pep[EG₁₈], 0.7% TNP^HER2pep[EG₁₈], and 1% TNP^HER2pep[EG₁₈]). 0.75% DiD was loaded to each nanoparticle as a fluorescent marker. Once tumor volume reached approximately 250 mm³, mice were treated with the nanoparticles via intravenous injections. After 24 h treatment, the mice were sacrificed, and organ tissues were dissected, collected, and analyzed for fluorescent nanoparticle tissue biodistribution. Fluorescence of each organ was read by IVIS Lumina II in vivo imager (PerkinElmer) at an emission wavelength of 640 nm and analyzed by Image J.

5.12. Nanoparticle tumor cell uptake assay

Following nanoparticle tissue biodistribution studies, the dissected tumors were disaggregated into single cells for analysis of nanoparticle tumor cell uptake. Each tumor chunk was placed in a 5 mL disaggregation solution (1 mg/mL collagenase type IV and 0.03 mg/mL DNase) in a 15 mL conical tube. The tumor-containing tubes were gently shaken in a 37 °C incubator for 45 min. Then, the solution was filtered through a 40 μm cell strainer (Cole-Parmer) into a 50 mL conical tube, and the cell strainers were washed with 5 mL PBS buffer. The collected cells were spun down by centrifugation using a centrifuge 5804R (Eppendorf). The cell pellets were washed three times and resuspended with 1 mL PBS, and the fluorescence signal was detected by a Guava easyCyte 8HT Flow Cytometer to analyze the nanoparticle tumor cell uptake.

5.13. In vivo efficacy and toxicity studies

The efficacy of Dox prodrug-loaded TNP^HER2pep was evaluated using the in vivo MMTV-neu transplantation mouse model [44]. Spontaneous tumors from MMTV-neu transgenic mice were first dissected and cryopreserved until they were used for transplantation. The tumors were transplanted to NOD-SCID female mice (n = 7 per group). Once tumors reached the size of ~150–200 mm³, mice were intravenously injected with PBS (Control), free dox (Dox), non-targeted Dox prodrug-loaded nanoparticles (NP_Dox), and Dox prodrug-loaded HER2 binding peptide presenting nanoparticles with various peptide density (0.2%, 0.35%, and 0.5%) and either EG8 or EG18 linker (0.2% TNP^HER2pep[EG₈]_Dox, 0.35% TNP^HER2pep[EG₈]_Dox, 0.5% TNP^HER2pep[EG₈]_Dox, 0.2% TNP^HER2pep[EG₁₈]_Dox, 0.35% TNP^HER2pep[EG₁₈]_Dox, and 0.5% TNP^HER2pep[EG₁₈]_Dox) at a dose of 3 mg/kg Dox equivalent on days 1, 3, 5, 7, and 9. Tumor volume and body weight were monitored throughout the study to evaluate antitumor efficacy and overall systemic toxicity, respectively. At day 24, the mice were sacrificed, and organs were dissected to be weighed in order to further evaluate overall systemic toxicity.