Surface engineered Fe₃O₄ nanomagnets for pH-responsive delivery of gemcitabine hydrochloride and in vivo tracking by radiolabeling

Abstract

Gemcitabine, a well-known nucleoside analogue, has shown tremendous potential against solid tumors. However, its clinical use is limited due to its poor biological half-life and low target to non-target ratio. In this regard, we have developed polyphosphate grafted Fe₃O₄ nanomagnets (PPNMs) for efficient delivery of gemcitabine hydrochloride (GEM). These nanocarriers are highly dispersible in physiological medium and possess sufficient surface charge (−25 mV) for electrostatic binding of positively charged GEM. The successful loading of GEM on PPNMs was evident from UV-visible absorption, FTIR spectroscopy and light scattering studies. The GEM loaded PPNMs (GEM-PPNMs) exhibited pH triggered release of the loaded drug, substantial cellular internalization, and higher toxicity towards human lung cancer (A549) and breast cancer (MCF-7) cell lines over pure drug. Further, the biodistribution of these nanocarriers was assessed by their tracking in a mouse model through radiolabeling with ⁶⁴Cu and ¹⁷⁷Lu. It has been observed that a radiolabeling yield of >90% can be achieved with PPNMs concentrations of 1 mg mL⁻¹ and 0.5 mg mL⁻¹ in 60 min for ⁶⁴Cu and ¹⁷⁷Lu, respectively, at room temperature. The radiolabeled PPNMs (⁶⁴Cu-PPNMs and ¹⁷⁷Lu-PPNMs) were highly stable during the study period in saline solution. Though the radiolabeled system exhibited higher uptake in the liver and spleen upon intravenous injection, a substantial uptake of the same was also found in the tumor. Specifically, the present study demonstrated the efficacy of PPNMs for delivery of GEM as well as their in vivo tracking by radiolabeling.

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Introduction

The advancement of innovative materials for further improvement of their existing biological applications is the main focus of researchers now-a-days worldwide. The design of nanomaterials with certain specific functional properties to achieve desired diagnostic and therapeutic efficacy is of prime importance.^1,2 This will refine the prevailing treatment procedure and help in eradicating the disease with ease. Starting from metal nanoparticles, metal oxide nanoparticles to metal and metal oxide hybrid nanoparticles are the choice of the decade for their simplicity in preparation and multifaceted applications. These nanoparticles provide a large surface area to play with and alter their overall activity as required time to time. Amid all this, Fe₃O₄ magnetic nanoparticles (MNPs) are typically significant for their exquisite magnetic properties which can change the course of the contemporary biomedical field. Because of their inimitable physicochemical properties and high biocompatibility nature, they can be used at cellular as well as subcellular level for numerous biological applications. Fe₃O₄ MNPs have been assessed for various biomedical applications such as cargo carriers for bioactive molecules (drugs, genes), next generation contrast agents for magnetic resonance imaging and localized heat mediated cancer cell killing via application of an external AC magnetic field.^3–7 Of all uses, MNPs are exhaustively used as carriers for delivery of different chemotherapeutic drugs via active and passive targeting routes.^8–10

Amongst chemotherapeutic agents, gemcitabine (2′,2′-difluorodeoxycytidine), a nucleoside analogue, has been used against a variety of human tumors.¹¹ It is a cytostatic drug which acts as an antimetabolite by inhibiting DNA replication. But the drawback of gemcitabine therapy lies in its rapid degradation which leads to low circulation time and the need for higher dosage. The use of higher dosage to achieve effective therapeutic efficacy leads to undesired side effects.¹² Further, gemcitabine has poor cell membrane permeability, and thus, it generally internalizes into the cells via nucleoside transporters due to its hydrophilic characteristics.¹³ Therefore, the expression of higher levels of nucleoside transporters is required in patients to achieve a substantial survival rate, which restricts its use in patients having lower levels of nucleoside transporters. There are very few studies reported in the literature on the delivery of gemcitabine using MNPs as a carrier, which could enhance its retention time and reduce its side effects.^14–16 Even though the use of MNPs as a drug carrier has many advantages, there are still some bottlenecks in their in vivo characteristics. Their efficacy was challenged many times due to recognition by our immune systems, mainly by the mononuclear phagocytic system (MPS) and reticuloendothelial system (RES) followed by clearance from the bloodstream as well as due to the presence of the blood–brain barrier. Thus, a strong competition exists between the distribution of MNPs in specific organs/tissues and their clearance mechanisms. The physicochemical property of MNPs affects the ultimate fate of the carriers upon administration in vivo.¹⁷ In order to increase the in vivo plasma half-life, different stealth coatings are used on the carriers.^18,19 Therefore, the addition of a suitable surface passivating agent defines the mode of action of Fe₃O₄ MNPs. Further, most of the studies are concerned with only in vitro level toxicity and cellular internalization. In order to achieve the actual potential of the nanocarrier, in vivo biodistribution studies are essential. Therefore, the tracking of Fe₃O₄ MNPs movement in an animal model has paramount importance in their biomedical applications. Especially, the labelling of MNPs with suitable radionuclides is an advanced technique for biodistribution studies as well as their in vivo tracking using various multimodal imaging techniques.^20,21

Herein, we have prepared negatively charged polyphosphate grafted Fe₃O₄ nanomagnets (PPNMs), and explored their efficacy for intracellular delivery of the anticancer drug, gemcitabine hydrochloride (GEM) through electrostatic interactions. These nanocarriers are highly dispersible in physiological medium, and GEM loaded PPNMs (GEM-PPNMs) exhibited pH dependent drug release characteristics, substantial cellular uptake and higher toxicity to human lung cancer (A549) and breast cancer (MCF-7) cell lines. Further, the biological activity of these nanocarriers was assessed by their tracking in a mouse model through radiolabeling with ⁶⁴Cu and ¹⁷⁷Lu. It is noteworthy to mention that the radiolabeled PPNMs were stable during the study period and substantial uptake of the same was also found in the tumor.

Experimental

Synthesis, structural characterization, drug interaction and cellular studies of PPNMs

PPNMs were synthesized through co-precipitation of ferrous and ferric ions (1 : 2 molar ratio) in the presence of ammonium hydroxide (25%) followed by the introduction of sodium tripolyphosphate (STTP) as a surface passivating agent.²² The structural characterization (phase identification, determination of shape, size and surface properties) and magnetic properties of PPNMs were studied by X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), dynamic light scattering (DLS), zetasizer and physical property measurement system, etc.

The interaction of GEM with PPNMs was explored by monitoring the changes in surface charge and absorbance. The drug loading efficiency was determined from UV-visible absorption spectroscopic studies at pH 4, 6 and 8 with different drug to particle ratios. The pH mediated release of the drug from GEM-PPNMs was achieved under reservoir (r)-sink (s) conditions at 37 °C. The cytotoxicity of GEM and GEM-PPNMs was examined against MCF-7 cells at five different concentrations of the drug ranging from 0.001 to 10 μM. The details of synthesis, structural characterization, drug loading and release, cytotoxicity, cellular uptake and cell death studies are provided in the ESI.†

Production of radionuclides (⁶⁴Cu and ¹⁷⁷Lu) and radiolabeling of PPNMs

⁶⁴Cu was produced via⁶⁴Zn(n,p)⁶⁴Cu by irradiation of the isotopically enriched (99.4% in ⁶⁴Zn) ZnO target. No-carrier added ⁶⁴Cu was extracted from the irradiated target in the form of [⁶⁴Cu]CuCl₂ solution in 0.01 M HCl following the procedure reported elsewhere.²³¹⁷⁷Lu was produced via the ¹⁷⁶Lu(n,γ)¹⁷⁷Lu route by direct neutron bombardment of the isotopically enriched (74.5% in ¹⁷⁶Lu) Lu₂O₃ target. The irradiated target was converted to [¹⁷⁷Lu]LuCl₃ solution in 0.01 M HCl as per the reported procedure.²⁴

Radiolabeling of PPNMs with ⁶⁴Cu and ¹⁷⁷Lu was performed using [⁶⁴Cu]CuCl₂ and [¹⁷⁷Lu]LuCl₃ radiochemical precursors, respectively. Briefly, 0.1 mL solution of [⁶⁴Cu]CuCl₂ (4 MBq of ⁶⁴Cu) or [¹⁷⁷Lu]LuCl₃ (20 MBq of ¹⁷⁷Lu) (both in 0.01 mL of HCl) was first added to the aqueous dispersion of PPNMs (0.2, 0.5, 1.0 and 2.0 mg in 1.0 mL of Milli Q water). The pH of the mixture was adjusted to ∼5.5 by addition of a few drops of 0.1 M NaHCO₃ solution. The total volume of the mixture was maintained at 2.0 mL using Milli Q water and the mixture was kept at room temperature under continuous stirring for adsorption of radionuclides onto the surface of PPNMs. Subsequently, the suspended radiolabeled PPNMs were magnetically separated by using a table-top magnet. For determination of percentage of radiolabeling, an aliquot (typically, 100 μL) of the clear supernatant was carefully withdrawn and its radioactivity was measured. The radioactivity of the same volume of ‘blank’ was also measured. The ‘blank’ solution was prepared by diluting 0.1 mL of [⁶⁴Cu]CuCl₂ (4 MBq of ⁶⁴Cu) or [¹⁷⁷Lu]LuCl₃ (20 MBq of ¹⁷⁷Lu) with 1.9 mL of Milli Q water, where no PPNMs were added. The percentage of radiolabeling yield was calculated using the following equation:

where ‘B’ and ‘R’ represent the background-corrected radioactivity of ‘blank’ and supernatant solution of the reaction mixture, respectively. The percentage of radiolabeling yield was determined for both ⁶⁴Cu- and ¹⁷⁷Lu-labeling as a function of concentration of PPNMs and time of incubation.

In vitro stability of radiolabeled PPNMs

The in vitro stability of ⁶⁴Cu- and ¹⁷⁷Lu-labeled PPNMs was investigated in normal saline and rat serum. In vitro stability in normal saline was explored by diluting 0.2 mL of [⁶⁴Cu]Cu-PPNMs and [¹⁷⁷Lu]Lu-PPNMs formulations with 1.8 mL of normal saline. The mixtures were kept at 37 °C for up to 2 days for the ⁶⁴Cu-labeled formulation and for 15 days for the ¹⁷⁷Lu-labeled formulation. The percentage of ⁶⁴Cu or ¹⁷⁷Lu activity leached out into the saline from radiolabeled formulations at different time intervals post-preparation was determined following the same procedure used for determination of radiolabeling yields. From this, the percentage radioactivity that remained associated with PPNMs as a function of time post-preparation was determined, which ascertains the in vitro stability of the formulations in saline.

In vitro stability in rat serum was studied by thoroughly mixing 0.2 mL of formulations with 1.8 mL of freshly isolated rat serum. The mixtures were kept at 37 °C for up to 2 days. The percentage radioactivity that remained associated with PPNMs as a function of time post-preparation was determined as in the previous case.

In vivo biodistribution studies of radiolabeled PPNMs

Biodistribution studies of the radiolabeled PPNMs formulations were performed in C57BL/6 mice bearing melanoma tumor as per the protocol approved by the Institutional Animal Ethics Committee of the Bhabha Atomic Research Centre, Mumbai, India.

Melanoma tumors were developed by injecting about 1 × 10⁶ melanoma cells suspended in 200 μL of PBS to the right thigh of C57BL/6 mice (weight: 20–25 g). The animals were monitored for development of tumors and the biodistribution studies were carried out once the tumor volume reached 150–200 mm³ (∼2 weeks after inoculation). An aliquot (0.1 mL) of the radiolabeled formulation (2–3 MBq) was injected intravenously into each tumor-bearing mouse. In the case of mice injected with ⁶⁴Cu-PPNMs, a group of 4 mice were sacrificed by CO₂ asphyxiation at 1, 4 and 24 h post injection (p.i.). Various organs/tissues were collected by thoroughly washing with saline (except blood) and dried. The radioactivity associated with them was determined using a flat-type NaI (Tl) scintillation counter. The percentage of injected radioactivity dose (% ID) in various organs/tissues was determined and provided as percentage injected radioactivity dose per gram (% ID g⁻¹) of organ/tissue. In the case of ¹⁷⁷Lu-PPNMs, biodistribution studies were performed at 1, 4, 24, 72 and 168 h p.i.

Results and discussion

The primary objective of the present study was exploration of the capability of negatively charged Fe₃O₄ nanomagnets for delivery of GEM and investigation of their in vivo biodistribution by radiolabeling. STTP was employed as a surface passivating agent due to its biocompatible nature and higher binding affinity towards Fe₃O₄via bidentate and tridentate bridging geometry. PPNMs were prepared as reported earlier by our group and well-characterized by XRD, TEM, FTIR, TGA and magnetic measurements.²² Here, the typical XRD pattern, FTIR spectrum, TEM image and magnetization plot of PPNMs are provided (Fig. 1) to confirm their successful phase formation and organic modifications. Briefly, the appearance of sharp and intense characteristic diffraction peaks corresponding to (220), (311), (400), (422), (511) and (440) planes in the XRD pattern of PPNMs (Fig. 1a) confirmed the formation of the highly crystalline single phase Fe₃O₄ nanostructure (JCPDS Card No. 88-0315). The characteristic stretching vibrational bands associated to P=O, PO₂ and PO₃ groups, and the P–O–P bridge are found in the FTIR spectrum of pure STTP (Fig. 1b).²² These modes are observed in the FTIR spectrum of PPNMs with a slight shift in their band position in the range of 700–1250 cm⁻¹, whereas they are absent in bare MNPs. Further, the intense band at ∼575 cm⁻¹ in PPNMs can be ascribed to the Fe–O stretching vibration of Fe₃O₄. The TEM image of PPNMs (Fig. 1c) reveals the formation of mostly spherical particles of size ∼10 nm and they show superparamagnetic behavior without hysteresis and remanence at 300 K. The maximum magnetization of PPNMs was found to be 51 emu g⁻¹ at 15 kOe.

Fig. 1 (a) XRD pattern, (b) FTIR spectrum, (c) TEM image and (d) magnetization plot of PPNMs. The FTIR spectra of STTP and bare MNPs are included in part (b) for comparative purpose. Bare MNPs (Fe₃O₄ nanoparticles without functionalization of STTP) were prepared by a similar method without using STTP during synthesis.

Colloidal stability of nanoparticles in physiological medium (PBS) is essential for their biological applications. Fig. 2 presents (a) DLS and (b) zeta-potential plots of PPNMs in physiological medium. The DLS plot (Fig. 2a) shows that PPNMs are highly dispersible in physiological medium (PBS, pH 7.4) and possess a mean intensity weighted hydrodynamic diameter of about 170 nm with a polydispersity index (PDI) of 0.25. The corresponding number weighted hydrodynamic diameter was found to be around 50 nm by using the DLS software. The zeta-potential analysis revealed that the surface charge of PPNMs in physiological medium was about −25 mV (Fig. 2b), whereas that of bare MNPs was −15 mV (Fig. S1, ESI†). The appearance of such a high negative surface charge on PPNPs is mainly due to the occurrence of phosphate moieties on their exterior, which is adequate for electrostatic stabilization of particles. Further, the hydrogen bonds between oxygen molecules of phosphate moieties and water provide additional colloidal stability to PPNMs. The good colloidal stability of PPNMs is also evident from the photograph of dispersion of PPNMs in physiological medium (inset of Fig. 2b) and the insignificant change in their hydrodynamic diameter in physiological medium with respect to time (Fig. S2, ESI†). Moreover, they should have lesser binding affinity towards macrophages, plasma proteins or other cell membranes being negatively charged.

Fig. 2 (a) DLS and (b) zeta-potential plots of PPNMs in physiological medium. The inset of part (b) shows the photograph of colloidal dispersion of PPNMs in physiological medium.

These negatively charged PPNMs were used for electrostatic conjugation of positively charged GEM (due to the protonation of the NH₂ group) as shown in Scheme 1. The loading efficiency of GEM was augmented by changing the ratio of drug to particles as well as pH of the medium, as shown in Table 1. A maximum loading efficiency of 23.7% was found at a drug to particle ratio of 1 : 40 at pH 6 from UV-visible absorption studies. Parsian et al. found a maximum loading efficiency of about 39% by interacting chitosan coated MNPs (2.5 mg mL⁻¹) with 22.5 μg mL⁻¹ of GEM.²⁵ Even though the maximum loading efficiency is quite low in the present study, the GEM loaded PPNMs exhibited higher cytotoxicity over pure GEM (discussed later). A typical UV-visible absorption spectrum of the supernatant solution (obtained after magnetic removal of GEM-PPNMs) along with standard curves are shown in Fig. S3 (ESI†). The surface charge of GEM-PPNMs in aqueous medium was −5 mV at pH 6, whereas that of PPNMs was −28 mV. This significant increase in surface charge from −28 mV to −5 mV could be described by neutralization of the negative charge through loading of GEM.¹⁴ The loading of GEM on PPNMs was further supported from FTIR and DLS analysis. Fig. 3 shows (a) the FTIR spectra of pure GEM and GEM-PPNMs and (b) the DLS plot of GEM-PPNMs showing the intensity weighted hydrodynamic diameter. The peak at 577 cm⁻¹ in the FTIR spectrum of GEM-PPNMs can be ascribed to the Fe–O stretching vibrational mode of Fe₃O₄.²² The FTIR spectrum of GEM showed bands at approximately 1645 and 1065 cm⁻¹ due to the bending modes of –NH/OH and stretching vibration of C–O, respectively.^26,27 The broad band that appeared in the 3000–3700 cm⁻¹ region can be ascribed to –NH/OH stretching vibrational modes.²⁷ Interestingly, these characteristic bands of GEM are also observed in the FTIR spectrum of GEM-PPNMs suggesting the loading of the drug onto the surface of PPNMs (absent in the FTIR spectrum of PPNMs). In addition, the increase in mean intensity weighted hydrodynamic diameter from 170 to 195 nm upon conjugation of GEM onto the surface of PPNMs further supports the successful loading of the drug. Moreover, the interaction of GEM-PPNMs with protein was explored utilizing BSA as a model protein and the observed results are shown in Table S1 (ESI†). The insignificant change in the surface charge of GEM-PPNMs in BSA solution suggested their good resistance towards protein interaction. The maximum drug loaded PPNMs sample was used for further studies.

Scheme 1 Pictorial representation of conjugation of GEM onto the surface of PPNMs.

Table 1 Loading efficiency of GEM obtained by varying the ratio of drug to particles as well as pH of the reaction medium

pH of the reaction medium	Amount of GEM (μg)	Amount of PPNMs (μg)	Drug to particle ratio	Loading efficiency (%)
6	10	50	1 : 5	2.3
	10	100	1 : 10	5.6
	10	200	1 : 20	10.5
	10	400	1 : 40	23.7
	10	800	1 : 80	21.6
4	10	400	1 : 40	19.0
6	10	400	1 : 40	23.7
8	10	400	1 : 40	21.9

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Fig. 3 (a) FTIR spectra of pure GEM, PPNMs and GEM-PPNMs and (b) DLS plot of GEM-PPNMs showing the intensity weighted hydrodynamic diameter.

Fig. 4 shows the pH responsive release of the drug from GEM-PPNMs under reservoir sink conditions at 37 °C. The drug release study was performed in a mild acidic medium as the pH of subcellular organelles like lysosomes and endosomes (late stage) is in the range of pH 3–5.²⁸ It was found that the loaded GEM was released in a pH dependent manner with higher release at lower pH. The amount of drug release over a period of 50 h was found to be 15, 45 and 80% at pH 7.4, 5 and 4, respectively. The short time release characteristic shows a linear relationship between GEM release and square root of time (t^1/2) as anticipated from the Higuchi drug release model (inset of Fig. 4). This indicates that the release of GEM from nanocarriers occurs in a diffusion-controlled manner.²⁹ It is worth mentioning that in a mild acidic medium (pH 5) the protonation of the phosphate group leads to a decrease in the electrostatic interaction between drug molecules and nanocarriers, thereby enhancing the release of the drug from the carrier. This was not observed in physiological medium (pH 7.4). At a still lower pH of 4, the drug release percentage was found to be further increased. Similar pH-responsive release of the drug from GEM loaded nanocarriers was observed by various research groups.^25,30–32 For instance, Parsian et al. reported only 8% release of GEM from chitosan coated MNPs at pH 7.2, whereas the release was 65.4% at pH 4.2.²⁵ Mottaghitalab et al. observed a faster release rate of GEM at pH 5 in comparison to pH 7.4 from the GEM loaded silk fibroin nanoparticles.³⁰ The observed pH mediated slow and sustained drug release profile in the present study suggested that the developed carriers can be utilized for intracellular delivery of GEM. Further, the quite slow release of GEM from the developed nanocarriers at pH 7.4 is highly beneficial for its stability in blood as the half-life of free GEM is very short (8–17 min) under physiological conditions.³³

Fig. 4 pH dependent release profile of the drug from GEM-PPNMs under reservoir sink conditions at 37 °C (the inset shows the linear relationship between drug release and square root of time). The standard deviation shown in the plot was determined by performing each experiment in triplicate.

Fig. 5 shows the viability of A549 and MCF-7 cells incubated in the medium containing GEM and GEM-PPNMs for 24 h and 48 h. From MTT assay, it has been found that both GEM and GEM-PPNMs induced dose-dependent toxicity against both A549 and MCF-7 cells upon incubation for 24 h and 48 h. The cytotoxicity of GEM and GEM-PPNMs is more prominent with 48 h incubation in both the cell lines. It is noteworthy to mention that the developed formulation has higher toxicity over pure GEM (toxicity data of GEM-PPNMs are statistically significant against the respective GEM group, p < 0.05), which is advantageous for therapeutic applications. The observed higher toxicity could be associated with the increase in cellular uptake of the drug by endocytosis of GEM-PPNMs.^25,32 In addition, the nanocarriers do not show toxicity to both A549 and MCF-7 cells up to a concentration of 200 μg mL⁻¹, suggesting their safe in vivo use (Fig. S4, ESI†).

Fig. 5 Viability of A549 and MCF-7 cells incubated in the medium containing GEM and GEM-PPNMs for (a) 24 h and (b) 48 h. Mean values were expressed as mean ± SEM for n = 3 in each experimental group. Statistical significance between GEM and GEM-PPNMs treatment groups was calculated using the Student's t-test, where * indicates p < 0.05.

Different staining techniques are commonly used to visualize the cellular processes by optical or fluorescence imaging of cells.^34–38 The cellular uptake of PPNMs was confirmed from the Prussian blue staining study in MCF-7 cells. From the vivid blue-coloured deposition (due to the formation of ferric ferrocyanide) in MCF-7 cells incubated with PPNMs (Fig. S5, ESI†), it can be deduced that a substantial amount of particles are successfully internalized into the cells as compared to their little cell surface attachment.³⁴ Acridine orange/ethidium bromide (AO/EB) fluorescence staining was further performed with A549 and MCF-7 cells to understand the cell death process after treating with GEM and GEM-PPNMs at a drug concentration of 2.5 μM for 48 h. Fig. 6 shows the fluorescent microscopic images of A549 and MCF-7 cells providing morphological evidence of apoptosis by AO/EB dual staining. The nuclei of cells having uniform chromatin with intact cell membranes (control or viable cells) are stained in green colour by AO, whereas those with loss of membrane integrity and with different stages of cell death are stained differently with EB. In general, the early apoptosis cells displayed a bright green region with yellowish green nuclear fragmentation, and membrane bubbles and apoptotic bodies outside, whereas the late apoptosis cells showed orange-yellow or red nuclei with condensed or fragmented chromatin.³⁹ The changes of colour from light orange to pale red in the present study suggested that most of the cells were dead via apoptosis mode due to the action of GEM and GEM-PPNMs on both the cancer cell lines. Thus, GEM-PPNMs may serve as a promising tool for intracellular delivery of drugs. Further, GEM resistance developed by some cancer cells due to the loss of certain human equilibrative nucleoside transporters (hENTs) can be overcome through its carrier mediated delivery as hENTs are not required for cellular uptake of the drug.⁴⁰ Being magnetic in nature, these nanocarriers can also be used as an effective heating source for hyperthermia therapy as well as a contrast agent in magnetic resonance imaging.^3,6 Moreover, these nanocarriers possess good magnetic field responsivity, and therefore, they can be accumulated at the site of interest by applying an external magnetic field.²⁵

Fig. 6 Fluorescent microscopic images of A549 and MCF-7 cells showing morphological evidence of apoptosis by AO/EB dual staining. The control cells show intact morphology, whereas a few apoptotic cells are observed in cells treated with GEM and GEM-PPNMs along with apoptotic bodies and fragmented nuclei.

Radiolabeling of Fe₃O₄ MNPs is a common method for monitoring the distribution of the nanoparticles in vivo.^20,21 We have carried out radiolabeling of PPNMs with ⁶⁴Cu²⁺ and ¹⁷⁷Lu³⁺ (Scheme 2) by exploiting the high coordinative affinity of polyphosphate moieties of PPNMs towards metal ions as a driving force for radiolabeling. It is noteworthy to mention that these polyphosphate moieties capture ⁶⁴Cu²⁺ and ¹⁷⁷Lu³⁺ ions from solution by forming chelate complexes like those of carboxylate ions and metal ions.⁴¹ The kinetics of radiolabeling with ⁶⁴Cu and ¹⁷⁷Lu using different concentrations of PPNMs is shown in Fig. 7(a) and (b), respectively. It has been observed that for ⁶⁴Cu, a radiolabeling yield of >90% can be achieved when a PPNMs concentration of 1 mg mL⁻¹ is used and the reactions are performed for 60 min at room temperature (Fig. 7a). On the other hand, the optimum condition for obtaining >90% radiolabeling with ¹⁷⁷Lu was found to be incubation of PPNMs at 0.5 mg mL⁻¹ concentration with ¹⁷⁷LuCl₃ for 60 min at room temperature (Fig. 7b). It has been found that the radiolabeling of PPNMs with ⁶⁴Cu and ¹⁷⁷Lu mostly follows a pseudo first order kinetic model. The typical plots showing radiolabeling of PPNMs with ⁶⁴Cu and ¹⁷⁷Lu (PPNMs concentration of 1 mg mL⁻¹) are provided in Fig. S6 (ESI†).

Scheme 2 Pictorial representation of radiolabeling of PPNMs with ⁶⁴Cu²⁺ and ¹⁷⁷Lu³⁺.

Fig. 7 Kinetics of radiolabeling of PPNMs with (a) ⁶⁴Cu and (b) ¹⁷⁷Lu using different concentrations of nanoparticles.

In vitro stability assessment of ⁶⁴Cu- and ¹⁷⁷Lu-labeled formulations showed that both the formulations retained their radiochemical stability in normal saline and rat serum up to the time of study mentioned in the experimental section. In all the cases, the percentage of radioactivity leached out from the radiolabeled formulations at the end of investigations was less than 5% (Fig. S7, ESI†). These results indicate the robustness of binding of ⁶⁴Cu and ¹⁷⁷Lu radioisotopes with PPNMs, which results in appreciably high radiochemical stability of the radiolabeled nanoformulations under physiological conditions.

After intravenous injection of ⁶⁴Cu- and ¹⁷⁷Lu-labeled PPNMs in C57BL/6 mice bearing melanoma tumor, in vivo biodistribution studies were performed by sacrificing the mice at different time points. The biodistribution patterns of ⁶⁴Cu- and ¹⁷⁷Lu-labeled PPNMs are shown in Fig. 8(a) and (b), respectively. It is observed that both ⁶⁴Cu- and ¹⁷⁷Lu-labelled PPNMs are primarily taken up by macrophages in different MPS organs, specifically liver and spleen, with immediate clearance from blood. The activity accumulated in the liver and spleen was found to decrease slowly with time. For example, the % ID g⁻¹ decrease in the liver (from ∼52.62% at 1 h to 30.08% at 168 h) and spleen (from 15.29% to 11.72% at 168 h) in ¹⁷⁷Lu-PPNMs treated mice indicates the clearance of PPNMs from the liver and spleen with time. However, a slower degradation rate was observed in spleen macrophages as compared to liver Kupffer cells possibly due to the presence of less iron storage proteins in spleen. The observed results match well with earlier reports on intravenously injected MNPs, which discussed their higher uptake in the liver and spleen.^42,43 A small fraction of PPNMs was also observed in the lungs and GIT, which slowly cleared with time. The radioactivity in the kidney showed that a minor portion of the radiolabeled PPNMs was excreted via urine. It is noteworthy to mention that the tumor uptake of PPNMs increases with time and a maximum uptake having 3.16% ID g⁻¹ was observed in ¹⁷⁷Lu-PPNMs treated mice at 24 h p.i., and this slowly decreased. Further, the retention of a substantial amount of radiolabeled PPNMs at the tumor site even at 168 h p.i. suggests their good potential as a nanocarrier for in vivo drug delivery. This is also required for radiotherapeutic usage. Moreover, the higher uptake of PPNMs in the liver can be useful for identifying liver cancer through MRI as higher uptake of PPNMs by Kupffer cells of the normal liver produces a dark contrast in T₂-weighted MRI, whereas the tumor sites lacking Kupffer cells and thereby phagocytized PPNMs appear as bright regions.⁴⁴

Fig. 8 Biodistribution patterns of (a) ⁶⁴Cu-PPNMs and (b) ¹⁷⁷Lu-PPNMs in C57BL/6 mice bearing melanoma tumor (a group of 4 mice were used for each time frame).

The ratios of target to nontarget uptake of the radiolabeled PPNMs were calculated and the results are shown in Table 2. In the case of ¹⁷⁷Lu-PPNMs, the tumor to blood ratio was found to increase from 0.78 at 1 h p.i. to 10.39 at 168 h p.i., and the tumor to liver ratio was found to increase from 0.04 to 0.08 during the same time period. The accretion of PPNMs at the tumor site can be further increased by conjugation of suitable targeting agents with PPNMs.⁴⁵ Specifically, the present study investigated the in vivo biodistribution of magnetic nanocarriers in a mouse model through radiolabeling and their efficacy for intracellular delivery of positively charged anticancer agents.

Table 2 Tumor/blood and tumor/liver ratio of ⁶⁴Cu- and ¹⁷⁷Lu-PPNMs at various time points post-administration in C57BL/6 mice bearing melanoma tumor

Time p.i.	1 (h)	4 (h)	24 (h)	72 (h)	168 (h)
^64Cu-PPNMs
Tumor/blood	0.62 ± 0.12	1.31 ± 0.16	1.96 ± 0.31	—	—
Tumor/liver	0.03 ± 0.01	0.06 ± 0.01	0.06 ± 0.12	—	—
^177Lu-PPNMs
Tumor/blood	0.78 ± 0.18	1.37 ± 0.17	2.86 ± 0.29	5.12 ± 0.48	10.39 ± 0.76
Tumor/liver	0.04 ± 0.01	0.06 ± 0.02	0.07 ± 0.02	0.08 ± 0.03	0.08 ± 0.10

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Conclusion

Anticancer drug, GEM was successfully loaded onto the negatively charged PPNMs through electrostatic interactions. GEM loaded PPNMs showed pH dependent release characteristics and exhibited higher toxicity towards MCF-7 and A549 cell lines over the pure drug. Further, the biodistribution of these nanocarriers was investigated in a mouse model by radiolabeling. The polyphosphate moieties on PPNMs capture ⁶⁴Cu²⁺ and ¹⁷⁷Lu³⁺ ions from solution by forming chelate complexes and this radiolabeling process follows a pseudo first order kinetic model. From in vivo studies, a substantial amount of radiolabeled PPNMs was found in the tumor. Therefore, the developed PPNMs can be used for delivery of GEM as well as in vivo tracking by radiolabeling.

Author contributions

Bijaideep Dutta: investigation, methodology, data curation, and writing – original draft. Sandeep B. Shelar: investigation, methodology, and data curation. K. C. Barick: project administration, writing – review & editing, supervision, conceptualization, and visualization. Priyalata Shetty: investigation, methodology, and validation. Rubel Chakravarty: investigation, validation, formal analysis, and writing – review & editing. Sudipta Chakraborty: formal analysis, visualization, and writing – review & editing. Swati Checker: investigation, methodology, and data curation. H. D. Sarma: investigation, methodology, and data curation. P. A. Hassan: project administration, writing – review & editing, supervision, and resources.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

The authors acknowledge Dr A. K. Tyagi, Director, Chemistry Group, and Dr S. Kannan, Director, Radiochemistry & Isotope Group, BARC for their constant support and encouragement. Thanks are due to Mr K. C. Jagadeesan and Dr S. V. Thakare, Radiopharmaceuticals Division, BARC for arranging irradiation of the targets in Dhruva and APSARA-U reactor facilities at BARC. The staff members of the Radiochemical Section, Radiopharmaceuticals Division, BARC are acknowledged for radiochemical processing of the irradiated targets. The authors are also thankful to IIT Bombay and TPD, BARC for TEM imaging and magnetic measurements, respectively.

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Footnote

† Electronic supplementary information (ESI) available: Materials, synthesis and structural characterization, drug loading and release studies, cytotoxicity, cellular uptake and cell death studies, zeta-potential, time dependent variations of hydrodynamic diameter, UV-visible absorption spectra, cell viability, Prussian blue staining, pseudo first order kinetic model fitting and percentage of radioactivity leached plots, and protein–particle interaction data (Fig. S1–S7 and Table S1). See DOI: https://doi.org/10.1039/d2ma00647b

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