1 Introduction

Brain diseases, such as central nervous system (CNS) disorders and brain cancers, are some of the most common and poorly treated diseases. Major challenges arises due to the lack of efficient technologies to deliver drugs across the BBB (Blood Brain Barrier). The selectivity of the blood–brain barrier in the passage of drugs inside the brain has been a challenge for many scientists. Several small molecules and macromolecules are investigated as effective therapeutic agents to treat various brain diseases. However, only molecules that are lipid soluble and also have a (molecular weight < 400 Da) can cross the BBB [1, 2]. Many promising classes of drugs, for example, nucleosides and derivatives established as anti-cancer, anti-viral, and anti-bacterial agents, have compromised CNS application because of the restricted BBB permeation [3]. Therefore, novel strategies are required for therapeutic targeting of drugs to brain tumours.

Current strategies to deliver therapeutic drugs in brain solely rely on proteins present in BBB and more recently strategies are researched which majorly includes delivery through active transporters in the BBB, brain permeability enhancers [4], non-invasive techniques such as FUS (focused ultrasound) and nasal route of drug administration. Nevertheless in spite of showing some promising outcome these techniques still faces some major limitations [5] in terms of size of the drug to be administered across BBB. The volume that can be intranasal administered is very small (25 μL) [6], which can limit the drug dose and the concentration of drug transported into brain which further diminishes the absorption time of drug inside. Ultimately the clinical application of these methods requires further investigation [7].

The genesis of drug-lipid is not serendipity, with nature having perfect examples such as the nucleoside-lipid hybrid conjugates or the nucleolipids. Lipidisation of the molecules leading to more lipophilicity has been certainly considered quite promising approach to make the drugs permeable across BBB, [8, 9]. There are FDA-approved CNS drugs wherein “Lipidisation” was used to convert a hydrophilic CNS drug that does not cross the BBB, to a lipid-soluble prodrug that does cross the BBB [10]. Nucleolipids were first isolated in bacterial strains as antimicrobials and since then have been widely reported synthetically by various groupsfor biomedical applications [11]. Some features such as self-assembling properties, especially in nano dimensions [12] have prompted researchers to develop nucleolipids as nano-drug delivery systems [13, 14].

The role of nucleolipid nanoparticles as drug delivery systems are comprehensively cited in literature and given importance for them being ideal candidates to be utilised as pharmaceutical formulations [15, 16]. Recently, rhodamine-conjugated nucleolipid nano-emulsion that internalized in the cells with liposomal co-localization was reported [17] addressing the efficiency of nucleolipid based nano drugs as biocomapatible drug delivery agents [18, 19]. It must be emphasized that almost all lipophilic prodrugs developed so far face poor solubility and bioavailability have therefore to be encapsulated into a nanocarrier, most often lipophilic particles, for improved pharmacokinetics. Nucleolipids are ideally considered for encapsulating hydrophobic drugs with an expected improvement of drug bioavailability for cancer cells and subsequent increased antitumor activity [20]. These systems are also useful in controlling drug payload release and protect drug from the surrounding environment. Wang et al. evaluated antitumor effect of 1% polysorbate-80 coated PBCA nanoparticles loaded with a nucleoside reductase inhibitor gemcitabine in vivo in C6 glioma cells [21].

We previously explored the role of di-C-15 ketalised nucleolipid for its brain tumor targeting in athymic xenograft mice having brain tumor using SPECT imaging [3]. After having encouraging resulted we assessed to address amphiphilic nature of the same nucleolipid molecule and look at its self-assembly as nanoparticles. These nanoparticles amplifies the lipidic-lipophilic effect, enhancing the brain uptake in BBB mice model and thus, paves the way for to penetrate BBB more effectively as compared to the standalone nucleolipid molecule. Herein we have established that nucleolipid based nanoparticles are stable, biocompatible and nontoxic hence can be efficient candidates for CNS based drug loading and biocompatible drug delivery agents.

2 Materials and Methods

All reagents and chemicals conformed to analytical grade and used as procured unless stated otherwise. The complete chemical synthesis, and characterization of nucleolipid molecule are reported [3]. In this contribution their formulation as nanoparticle has been carried out using dichloromethane (DCM), methotrexate (purity ≥ 99%), and for dialysis in water, cellulose dialysis membrane (3–5 kDa) were procured from Sigma-Aldrich (St. Louis, MO).

For the in vitro studies, the reagents, buffers (Tris/ PBS), triton X-100, Sulforhodamine B monosodium salt (SRB, dye content 75%) dye, Hanks’ balanced salt solution (HBS) were procured from Sigma-Aldrich.

2.1 Ethical approval

All animal experiments were performed following the guidelines of the CPCSEA, Government of India, New Delhi. Animal handling and experimentation were approved by the Institutional Animal Ethics Committee (IAEC) of INMAS (Reg. no.8/G0/Rbi/S/99/CPCSEA;09–03-2014).

2.2 Formation of nanoparticle

Nanoparticles NLDPU-DTPA-Np were prepared through nanoprecipitation while using dropping method in aqueous media with slight modification [22]. This technique is based on intrafacial deposition due to displacement of one solvent with the non solvent thus provides clear results. Briefly, 10 mg of nucleolipid- DPU-DTPA,(the synthesis of DPU-DTPA is reported elsewhere) [3] was solubilised in 1 mL of dichloromethane at room temperature.100 µL of this was added drop wise to 10 mL of distilled water under constant magnetic stirring to obtain nanoemulsion. We did not use any amphiphilic surfactant in the preparation as nucleolipids by virtue of their structure have lipid chain which mimics phospholipid membrane and has ability to form nanobodies when dissolved in aqueous medium. The nanoemulsion was then placed in ultrasonic water bath for briefly 20 min at 40 °C. Residual Solvent (DCM) was removed by slow evaporation under reduced pressure using rotavapor and the total volume of resultant mixture containing nanoparticle was adjusted to 1 mL.

2.3 Scanning electron microscopy (SEM)

The opalescent nanoemulsion was filtered using 200 nm filter and used for the determination of the hydrodynamic diameter and zeta potential of the nanoparticles using the principle of dynamic light scattering carried on Malvern Zetasizer NanoZS, UK. Triplicate measurements were performed. The morphology was analyzed by Scanning Electron Microscope (M/s Hitachi, H-7500, Japan) at 25 °C.

2.4 Treatment of HEK 293 Cells with NLDPU-DTPA-Np

Human embryonic kidney (HEK) cell lines were procured from National Centre for Cell Science (NCCS) Pune, India. The cell line was cultured in high glucose DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics: 1% (v/v) penicillin–streptomycin. The cell line was maintained in humidified 5% CO2 incubator at 37 °C. Cells were sub-cultured routinely twice a week using 0.05% trypsin in 0.02% EDTA [23]. Cells were seeded at cellular density of 5000 cells per well in 96 well microtiter plate and treated with varying concentration of 99mTc- NLDPU-DTPA-Np (0.01 μM-1000 μM) for different time intervals (24, 48 and 72 h). After the intended time, the wells were emptied and the cells were fixed with 100 μL of ice cold 10% (w/v) trichloroacetic acid (TCA) at 4 °C for 1 h. After washing of the well gently with running water followed air-drying, 100 μL of 0.05% SRB solution was added for 30 min for staining.

2.5 Haemolytic toxicity assay

Time-dependent haemolytic toxicity was assessed by incubating red blood cells (RBC) with 5 mg/mL 99mTc-NLDPU-DTPA-Np for different time points (0.5 h, 1 h, 2 h, and 4 h). The quantification of disintegrated haem was done by measuring the absorbance at 540 nm using Synergy 2 Multi-mode reader (M/S Biotek Instruments, USA). Positive and negative control samples contained Triton X-100 (2% v/v) and saline respectively. Triton X-100 sample was regarded as 100% haemolysis.

The unbound dye was washed (4 ×) with 1% (v/v) acetic acid. SRB-bound cell protein was solubilised using 200 μL of 10 mM Tris base solution (pH 10.5) and quantified fluorometrically (λexcitation 280 nm and λemission 400 nm) on Biotek Synergy H4 hybrid multiplate reader. HBS buffer treated cells served as control. Surviving fraction was calculated and plotted for the concentration range 1 nM–1000 μM as a function of time.

2.6 Nanoparticle methotraxate loading

The drug loading and encapsulating efficiency was evaluated using the anticancer drug, methotrexate (MTX). The method of preparation of MTX–loaded nanoparticles was similar as Sect. (2.1), except nano-encapsulation was carried in soft illumination. MTX was dissolved in DMSO (1 mL) and mixed with nanoemulsion of nucleolipid nanoparticles. The resulting mixture was dialysed under dark conditions for 24 h to remove unloaded drug as well as the undesired solvent. To further comprehend that the unloaded nanoparticles are free from MTX loaded nanoparticles, UV analysis was done.

To quantify the amount of MTX encapsulated in the nucleolipidic nanoparticles,they were dissolved in 1–2 mL of DMSO so that ruptured nanoparticles release the entrapped drug. The resulting solution was further analysed through UV–Vis Synergy 2 Multi-mode reader (M/S BioTek Instruments, USA). At 180–700 nm the absorbance at λmax 375 nm was recorded and compared with the standard calibration curve of MTX (0.1–1.0 mg/mL). Triplicate experiments were performed and corrected for background.

Finally the Encapsulation efficiency and the drug loading content were calculated using the following formula

$$\% {\text{Encapsulation}}\;{\text{efficiency}} = \frac{{{\text{Amount}}\;{\text{of}}\;{\text{encapsulated}}\;{\text{drug}}}}{{{\text{Amount}} {\text{of}} {\text{drug }}}} \times 100$$
$$\% {\text{Drug}}\;{\text{loading }} = \frac{{{\text{Amount}}\;{\text{of}}\;{\text{encapsulated}} {\text{drug}}}}{{{\text{Amount}}\;{\text{of}}\;{\text{Nanoparticle}} }} \times 100$$

2.7 In-vitro drug release kinetics and modelling

The protocol for drug release kinetics and the mathematical modelling was followed as reported in Ref. [24]. Dialysis method was used to determine the drug release kinetics for 24 h or 100% release, whichever was earlier. Briefly, 1 mg of MTX loaded nanoparticles was placed in a regenerated cellulose dialysis bag (MWCO of 300 Da) dispersed in phosphate buffer which served as the dialysis medium. The relative volume of dialysis buffer and was 100 fold as compared to the nanoparticle suspension. The release of methotrexate into the dialysis medium was monitored by measuring absorbance of 2 mL aliquot of dialysate at 375 nm. On withdrawing the 2 mL aliquot, the dialysis medium was replenished with equal volume to maintain constant sink conditions.

2.8 In vivo studies

2.8.1 Animal model

All animal experiments and study protocols were as per the approved protocols by the INMAS institutional animal ethical committee (IAEC).The experimental animal facility (EAF) at INMAS provided female BALB/C mice (5 − 6 weeks old and weighing 23 ± 2 g) and New Zealand albino rabbits (weighing 2–-3 kg). The animals were housed comfortably at 21 ± 2 °C in sterile and pathogen-free conditions with a 12 h day and night cycle and were provided with standard food and water ad libitum.

The Board of Radiation and Isotope Technology (BRIT), Delhi, India supplied 99mTc-pertechnetate. As 99mTc is radioactive having gamma emissions with energy of 140 keV, appropriate precautionary measures were adopted while experimentation. The radioactive counts were recorded on 99mTc-calibrated gamma well-type counter (Caprac R Capintec, USA).

2.8.1.1 Radiolabelling and stability

The NLDPU-DTPA-Np was radiolabelled with 99mTc in presence of stannous tartarate as reducing agent using standard protocol with minor modifications [3]. The conditions optimized for achieving maximum radiolabelling efficiency were confirmed by ascending thin layer chromatography (TLC) on silica gel-coated fibre sheets (ITLC-SG: Gelman Science Inc., Ann Arbor, MI, USA). The strips were cut in parts and radioactive counts for each part were measured in gamma counter. Further analysis of the strips was carried as reported in [3] to calculate radiolabelling efficiency.

2.8.1.2 Serum stability

The stability was assessed by incubating 99mTc-NLDPU-DTPA-Np with freshly collected human serum devoid of any clotting factors after 0.22µ filtration. After pre-defined incubation of PBS-diluted serum (50% v/v, pH7.4)) with 0.2 mL of 99mTc-NLNP (12 MBq, 1.6 mg), the samples were spotted on ITLC-SG and analysed as above 2.3.1.

2.8.1.3 Trans-metalation stability

Trans-metalation stability was assessed using cysteine challenge experiment in which 0.5 mL of 99mTc-NLDPU-DTPA-Np (0.3 MBq, 0.5 mg in PBS) was incubated with varying concentration of cysteine solution (25, 50, and 100 mM in 1 mL water). After incubation of 1 h at 37 °C, aliquots were spotted on a ITLC-SG strip, developed in 0.1 M PBS and counted in gamma counter. The radiolabeled cysteine had an Rf of 0.81 whereas the 99mTc-NLDPU-DTPA-Np remained at the bottom.

2.8.2 Pharmacokinetic and bio distribution study

The biodistribution study was performed in normal and mannitol administered BALB/c mice in a time-dependent profile similar to the procedure reported [25]. Mannitol (30%), a hypertonic osmolytic permealiser for blood brain barrier (BBB) was administered 15 min before injecting 99mTc- NLDPU-DTPA-Np. For each time point (viz., 5 min, 15 min, 60 min and 120 min), triplicate sets of mice were injected with 20 μL of 99mTc- NLDPU-DTPA-Np (11.1 ± 2.7 MBq). At intended time post-injection, mice were sacrificed through the cardiac puncture, and different organs and tissues were excised. The radioactivity in the organs was measured in the gamma well counter. Uptake of 99mTc-NLDPU-DTPA-Np in each tissue was expressed as a percentage injected dose per gram of the tissue (% ID/g) and corrected for 99mTc decay.

3 Result and discussion

After having, encouraging results of nucleolipids ability to cross BBB and brain targeting for early diagnostics from our previous work [3]; In this research we attempted to magnify the effect of lipidization by formation of nanoassemblies of the nucleolipid molecule. The aim of the study was to give more insight in the nucleolipid by converting them into nanoparticles as in the nano form they have more improved pharmacokinetics as compared to the standlone nucleolipid. Our idea got further motivation from the recently reported nucleolipid nanoemulsion to cross plasma membranes [17]. The non-toxicity and the self-assembly properties of the nucleolipids render them ideal amphiphilic adjuvant for encapsulating hydrophobic anticancer drugs such as methotrexate.

3.1 Formulation of nucleolipid nanoparticles

The design and synthesis of the nucleolipid molecule (DPU-OH, DPU-DTPA) is described elsewhere [3]. The nucleolipid had three components (a) nucleoside, (b) lipid chain, and (c) chelator, DTPA) for loading of 99mTc (Fig. 1). The nanoparticle were prepared by a nano-precipitation method in aqueous media. The resultant nanoparticle emulsion was opalescent in nature (Fig. S3).

Fig. 1
figure 1

Construct of nucleolipid nanoparticles

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3.1.1 Characterization of NLDPU-DTPA-Np

We observed mean particle size of NLDPU-DTPA-Np 113 nm with poly-dispersity index of 0.25 which exhibited a narrow size distribution. The particle size is optimized to smaller size (d < 200 nm) as to have long circulation time in the blood. Zeta potential or the electrokinetic potential at the shear plane is an important index for the stability of nanoparticles by indicating the surface charge and net repulsion of the particles [26]. The value of zeta potential was found to be − 22.0 mV, indicating moderate stability at room temperature. The negative value of zeta potential was due to presence of carboxylic acid in DTPA (Fig. 2). The SEM image of the NLDPU-DTPA-Np depicted spherical and uniform morphology (Fig. 2c), (Figs. S4–S7).

Fig. 2
figure 2

Dynamic Light scattering data (a) and Zeta Potential graph (b) to evaluate average particle size of the formed nanoparticles. The distribution in fig (a) shows an average particle size of 113 with polydispersity index of 0.25, indicating a narrow size distribution (fig b). SEM image of nanoparticles acquired at z 200.0 kV Magnification (c) showing spherical morphology of the nanoparticles

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3.1.2 Stability of 99mTc-NLDPU-DTPA-Np

The stability of nanoparticles was our major consideration in all steps of preparation, from processing steps to storage. The particle size was assessed as function of pH and time over the period of 60 days. The stability studies were carried, and size measurements were recorded (Fig. 3b). For this Study DPU-OH the original nucleolipid and DPU-DTPA (modified with chelator) was assessed parallely in order to check if there is any change in the nature of nanoparticles due to its chemical modification, as appending DTPA was crucial in order to radiolabel the nanoparticles with radioactive metal (99mTc). Initially the physical appearance of nanoparticles stored at 24 °C for 1, 2, and 3 months was evaluated. At the end of 1 month, nanoparticles stored at 24 °C were stable but a change in the color and viscosity was observed after 30 days which led to discontinuation of the stability studies at 24 °C after 1 month. For DPU-OH, and DPU–DTPA there is slight change in the particle size and size distribution after few days, stored at 24 °C this confers that NLDPU-DTPA-Np are more stable over NLDPU-OH-Np. The Chemical stability was accessed at different pH and observation was recorded. The nanoparticles were found to be remaining stable as they show similar particle size for about 2 weeks. A slight degradation was observed at pH 3 which is in accordance with the fact that nucleolipids are susceptible to acidic pH due to their ketal linkages present in their structure (Fig. 3a).

Fig. 3
figure 3

Stability data of formed nanoparticles a effect of pH on nanoparticles NLDPU-DTPA-Np are more stable over NLDPU-OH-Np. b stability of NLDPU-DTPA-Np are more stable over NLDPU-OH-Np in their size over the period of days (60)

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3.2 In vitro parameters

The NLDPU-DTPA-Np is designed eventually for biomedical applications, particularly drug delivery. Since intravenous is the preferred administration route, and the nanoparticles directly passes into the blood (1) toxicity effect on erythrocytes (red blood cells) and (2) toxicity effect on cells were evaluated.

3.2.1 Toxicity assessment

  1. (a)

    Haemolytic toxicity.

Due to high lipophilicity, NLDPU-DTPA-Np can exert toxic effects on erythrocytes. The analysis is based on the principle that a haemo-toxic compound ruptures the erythrocytes, and release red colored haem in the solution which can be spectroscopically quantified (λabs 540 nm). The NLDPU-DTPA-Np showed only 2.33% erythrocyte destruction until 4 h, suggesting no appreciable haemolytic effect, and therefore ensuring a discernible compatibility with blood cells (Fig. 4a).

Fig. 4
figure 4

a Haemolysis data for NLDPU-DTPA-Np in comparison to saline (negative control) and Triton X-100 (positive control) treated RBC. The results highlight the non-toxic effect of NLDPU-DTPA-Np on RBC. b SRB cytotoxicity assay on HEK cell line. At 72 h with extended treatment of NLDPU-DTPA-Np, 76 ± 0.3% cells survived, indicating that the particles are non-toxic

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  1. (b)

    Cellular toxicity.

The SRB based cytotoxic assay revealed no cellular cytotoxicity of NLDPU-DTPA-Np in the concentration range of 1 nM to 1 mM on HEK-293 cell line. A slight decline in surviving fraction was observed as concentration was increased. At 72 h of NLDPU-DTPA-Np extended treatment, more than76 ± 0.3% surviving fraction at 1 mM was observed. IC50 value (50% cell lysis) was not observed for NLDPU-DTPA-Np even at concentration 1000 µM for all incubation times, indicating the non-toxic nature of NLDPU-DTPA-Np (Fig. 4b).

3.2.2 Drug loading and encapsulation

The drug loading efficiency of NLDPU-DTPA-Np was assessed for the anticancer drug, methotrexate using U.V. spectroscopy. The standard calibration curve for methotrexate at λmax375 nm with correlation coefficient r2 0.999 was used for its estimation (Fig. 3).The loading efficiency of lipid soluble drug can be affected by the lipid concentration, type of lipid used (saturated/ unsaturated) and cholesterol content in the formulation [27]. In the present case, the hydrophobic nature of NLDPU-DTPA-Np would facilitate the encapsulation of methotrexate. The encapsulation of the drug, methotrexate was followed using absorbance studies. The respective values of the encapsulation efficiency and the drug loading of MTX in NLDPU-DTPA-Np were found to be 68 ± 2.75% and 22 ± 1.8% respectively.

3.2.3 Drug release and release modelling

The mechanism of release of anticancer drug methotrexate (MTX) from the MTX-loaded nanoparticles (NLDPU-DTPA-Np-MTX) could be elucidated through mathematical modelling of controlled release systems. Therefore, the results obtained from in vitro drug release studies was generated to various kinetic equations, vis-a-vis the zero-order, first-order, Higuchi, and Korsmeyer-Peppas mathematical models, in order to have a better understanding for the underlying mechanism of release. The drug release of NLDPU-DTPA-Np was evaluated through aliquot analysis of dialysate for MTX by UV spectroscopy. Three characteristic absorption maxima of MTX at 258, 302 and 375 nm were obtained. The absorbance maxima at λ = 375 nm was used for calibration curve. The correlation coefficient of 0.999 was obtained after fitting the regression line (Fig. 5).The in vitro release profile of methotrexate from NLDPU-DTPA-Np is shown in Fig. 5. Non-encapsulated methotrexate released completely from the dialysis bag into the dialysis sink within 8 h. When encapsulated, the biphasic release was observed with 100% release occurring in a span of 24 h. The initial burst release occurred with 50% release within 4 h. After the early release, the drug discharged slowed down to almost zero-order rate, presenting a typical sustained and prolonged drug release. Such a phenomenon is well reported and occurs due to the poorly surface adsorbed particles of the drug with later release predominantly occurring through diffusion for the encapsulated drug [28].

Fig. 5
figure 5

a Drug release kinetics of NLDPU-DTPA-Np entrapping methotrexate with comparison with free methotrexate. The free MTX released 100% within 8 h whereas 100% release of encapsulated MTX occurred in a span of 24 h. b Using the mathematical models Higuichi model the release constant was found to be 20.79. c Korsemeyer Peppas model where the value of slope indicates the type of diffusion release

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3.3 In-vivo studies

3.3.1 Radiolabelling and serum stability studies

The nanoparticles was radiolabeled with 99mTc. 99mTc is a gamma emitting radionuclide, which can be counted using gamma counter or can be tracked in SPECT imaging. The radiolabelling with 99mTc as per the standard previously reported procedure [3]. Optimal radiolabelling was achieved at room temperature with 99mTc-pertechnetate (30 ± 10 MBq) using 0.1 mg/mL stannous tartrate. With more than 98 ± 0.75% radiolabelling efficiency (Fig. 6a) and labelling efficiency was further found intact with incubation time with reducing agent (Fig. 6b) the 99mT NLDPU-DTPA-Np was tested for in-vivo stability in serum and were stable up to 24 h when incubated with serum with 90% of the complex remaining intact, implying the suitability for in vivo application (Fig. 7a). The results of cysteine challenge studies carried to assess the propensity for transmetallation with cysteine residues present in proteins were also in favor with nearly 8% of the complex getting dissociated after incubation with 100 mM of cysteine (Fig. 7b).

Fig. 6
figure 6

Radiolabelling optimisation a) effect of concentration of reducing agent, b) effect of incubation time

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Fig. 7
figure 7

a) Serum stability assay b)  Cysteine challenge studies which indicates very low instability of 99mTc-NLDPU-DTPA-Np

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3.3.2 Pharmacokinetic and bio distribution study

The 99mTc- NLDPU-DTPA-Np were intravenously injected through ear vein into healthy rabbits to generate their blood clearance profile. A comparative evaluation revealed a gradual clearance of these nanoparticles from systemic circulation over a period of 24 h, p.i., exhibiting a bi-exponential pattern (Fig. S1). The enhanced blood circulation time and clearance of 99mTc-NLDPU-DTPA-Np may be explained as also reported for other lipidic nanoparticles [29]. The 99mTc-NLDPU-DTPA-Np cleared rapidly from the blood with 50% total blood activity remaining at t1/2 ≈ 20 min. After having blood kinetics profile being evaluated which substantiated the ground for further biological evaluation we used mice as animal model for determining brain uptake through biodistribution.

Bio-distribution studies were carried out in two different animals (mice) to compare the uptake of radiolabeled nanoparticles (99mTc-NLDPU-DTPA-Np) in the brain of two animals and also study the variation in the pattern of distribution. Octanol, is widely used as osmotic permealizer and can enhance the permeability of various membranes including mucosa, skin and the BBB [30]. We evaluated the extent of BBB penetration capability of 99mTc-NLDPU-DTPA-Np in normal mice having intact blood brain barrier in comparison to the mannitol treated mice having impaired blood brain barrier. The mannitol treated mice serves as the proof of concept for real time imaging of nanoparticles in tumorigenic mice having disrupted BBB. Octanol treated BBB opening technique in mice can be utilized to study the biology of the BBB and improve the delivery of various therapeutic agents to the brain. In normal mice 99mTc-NLDPU-DTPA-Np showed an uptake of 1.6% I.D at 5 min in comparison to 1.78% I.D in mannitol treated mice in similar experimental conditions indicating the potential of the 99mTc-NLDPU-DTPA-Np to cross the BBB \(\left( {\frac{{{\text{Brain}} \;{\text{uptake}} \left( {{\text{normal}}\;{\text{mice}} } \right)}}{{{\text{Brain}}\;{\text{uptake}} \;\left( {{\text{BBB}}\;{\text{compromised}}} \right)}} = \, 0.91} \right)\) (Fig. 8a). The blood to brain ratio (0.75) is also calculated in order to distinguish the effective brain uptake of 99mTc-NLDPU-DTPA-Np in brain. Parallel to this biodistribution was assessed in nornmal mice for its quantitative uptake in different organs. The study showed nanoparticles haves both hepatic and renal clearance (owing to the hydrophilic nature of DTPA chelator). The low activity in stomach indicated further stability of the nano particles [31] (Fig. 8,b).

Fig. 8
figure 8

a Bio distribution study of brain in normal mice for 99mTc-NLDPU-DTPA-Np showing brain uptake (1.6% ID), versus Brain Uptake values mannitol treated mice, data presented as% ID/g for all organs (mean ± SD), n = 3, There is no statistical difference among for values in normal mice and octanol treated mice (P = 0.4). b Comparative evaluation of uptake values for.99mTc-NLDPU-DTPA-Np in different organs at different time points, data presented as% ID/g for all organs (mean ± SD), n = 3

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4 Conclusion

Brain targeting and drug delivery is triggering issue because of the discriminatory function of the BBB. There is no impeccable technique for the safe delivery of anticancer agents into the brain. Biocompatible nanoscale multifunctional nanomedicines offer trinity for targeting, imaging, and therapy for CNS disorders namely brain tumor. In this work, the authors have extended the application of the previously reported nucleolipid as nano-particles preparation and improved brain targeting capabilities due to the lipidization effect of nucleolipids supramolecular assembly. The nucleolipid-nanoparticles (99mTc-NLDPU-DTPA-Np) has been evaluated for its haemolytic cytocompatibility, radiolabelling, and brain permeability efficiency across blood–brain barrier. Parallel, the nanoparticles has also been assessed for drug encapsulation and release kinetics in vitro. The results indicate the suitability of the nucleolipid nanoparticles in the development of drug delivery systems. As the design of the nucleolipid-nanoparticles incorporated a chelator, these particles can be radiolabelled with futuristic application in multimodal imaging peptide receptor radionuclide therapy (PRRT).