A method to improve quantitative radiotracing‐based analysis of the in vivo biodistribution of drug carriers

Abstract Biodistribution studies are essential in drug carrier design and translation, and radiotracing provides a sensitive quantitation for this purpose. Yet, for biodegradable formulations, small amounts of free‐label signal may arise prior to or immediately after injection in animal models, causing potentially confounding biodistribution results. In this study, we refined a method to overcome this obstacle. First, we verified free signal generation in animal samples and then, mimicking it in a controllable setting, we injected mice intravenously with a radiolabeled drug carrier formulation (125I‐antibody/3DNA) containing a known amount of free radiolabel (125I), or free 125I alone as a control. Corrected biodistribution data were obtained by separating the free radiolabel from blood and organs postmortem, using trichloroacetic acid precipitation, and subtracting the confounding signal from each tissue measurement. Control free 125I‐radiolabel was detected at ≥85% accuracy in blood and tissues, validating the method. It biodistributed very heterogeneously among organs (0.6–39 %ID/g), indicating that any free 125I generated in the body or present in an injected formulation cannot be simply corrected to the free‐label fraction in the original preparation, but the free label must be empirically measured in each organ. Application of this method to the biodistribution of 125I‐antibody/3DNA, including formulations directed to endothelial target ICAM‐1, showed accurate classification of free 125I species in blood and tissues. In addition, this technique rendered data on the in vivo degradation of the traced agents over time. Thus, this is a valuable technique to obtain accurate measurements of biodistribution using 125I and possibly other radiotracers.

I and possibly other radiotracers.

K E Y W O R D S
biodistribution data correction, degradation, drug delivery carriers, free label, in vivo biodistribution, radiotracing, trichloroacetic acid precipitation
Radiotracing methods are interesting because they provide high sensitivity. 6,7 They require labeling agents with a radioisotope or substitutive incorporation during synthesis. 23,24 Agents of known specific activity are administered in vivo, followed by collection of tissues for ex vivo measurements. As an example, 125 I can be used to label peptides, proteins, nucleic acids, nanoscale materials, and so on, enabling their quantification in a γ-counter. 6 While the inherent risks of using γ emitters must be balanced against the convenience of analysis, ex vivo γ-radiotracing provides standardized quantitation without tissue matrix interference, complex processing, routine calibrations, standard curves or quality control samples, while providing sub-parts-per-billion sensitivity and multiplexing capacity. 6,23,24 Signal loss due to radioactive decay is known for each radioisotope and can be quantitated. Numerous publications validate the use of ex vivo γ radiotracing for biodistribution studies of drugs and their carriers. [25][26][27][28][29][30] Yet, like other methods, radiotracing may suffer from a noise signal that originates from the presence of free label. Free label, for example, radio-iodine and other radiohalogens, may arise by detachment from DDSs upon interaction with the body or degradation of biodegradable DDS components. Polymers, targeting antibodies, and/or cargoes could be labeled with a radioisotope and their body degradation could release their monomolecular building blocks linked to the respective radiolabel (herein called "free label" for simplicity).
Free label could also be released prior to administration, depending on the DDS shelf-life. Due to the high sensitivity of radiotracing, free label can confound biodistribution results due to their different accumulation profile versus a DDS. Since this cannot be predicted, organspecific corrections of the measurements are essential. Modification of the radiotracing biodistribution method is necessary such that the true signal originating from the label attached to the DDS formulation can be distinguished from the signal originating from any possible free label.
Here, we investigated whether precipitation with trichloroacetic acid (TCA), a method used in vitro to separate free radiolabel from radiolabeled biological polymers, such as proteins or nucleic acids, 31,32 could be applied to precisely elucidate the in vivo biodistribution of a DDS. Results demonstrate that small amounts of free label do generate in the biological milieu and that this method can be used to identify free 125 I present in blood and organs, providing a valuable means for correction of free radiolabel present in samples that are used for biodistribution studies. Small amounts of tracer may arise from radiolabeled DDS, which could confound biodistribution studies. To illustrate this, we used a DDS called 3DNA®, a DNA construct assembled from single-stranded polynucleotides designed to hybridize into modules and layers, via sequence complementarity 33 (section 4), resulting in a drug nanocarrier. [33][34][35][36] The 3DNA formulation used here has been recently characterized 37 and has 170 ± 7 nm hydrodynamic diameter, −19 ± 0.6 mV ζ-potential, and 0.22 ± 0.003 polydispersity index (PDI). To mimic formulations used in targeting strategies, these nanocarriers were coupled to an antibody (Ab) labeled with 125 I ( 125 I-Ab) and modified with an oligonucleotide complementary to 3DNA outer arms, 35,37,38 which increased the average hydrodynamic diameter to 181 ± 5 nm and decreased the ζ-potential to −42 ± 0.5 mV, with similar PDI of 0.23 ± 0.019.
We tested the possible release of free 125 I from 125 I-Ab/3DNA ( Figure 1) by incubating this formulation with injection buffer (preadministration simulation), mouse blood, liver or kidney homogenates (post-administration simulation). Free 125 I was separated by adapting an established method that employs TCA, an agent used to precipitate biological polymers (proteins, nucleic acids, and polysaccharides to some extent), allowing easy separation of free label after centrifugation. 31,32 Release of small amounts of free 125 I was detected in pre-and post-administration simulations, ranging from 2% to 11% (at 60 min) of the total signal. Most of the label release occurred in the first 5 min (4, 4, 7% release for buffer, blood, and liver), indicating a fast event. Free-label release was similar in buffer and blood for early time points, and increased in blood by 60 min, as expected. Also as expected, release was greater in the liver and kidney homogenates versus the blood and buffer. This highlights that: (a) there is low (≤11) release of free label from 125 I-Ab/3DNA in physiological conditions, validating that this radiolabel is sufficiently stable to trace biodistribution within the selected time range; and (b) there is some degree of fast label release pre-and postadministration (4% in buffer and blood at 5 min). Thus, there is a need to trace this confounding signal, for which we compared the biodistribution upon i.v. administration in mice of 125 I-Ab/3DNA containing known amounts of free 125 I (a controlled situation) versus free 125 I alone, measured prior to and after TCA precipitation ( Figure S1).

| Biodistribution of i.v. administered free 125 I radiolabel
We determined the biodistribution of free 125 I injected i.v. in mice. As expected for such a small molecule, we observed fast blood clearance ( Figure 2 (Table S1). Free 125 I accumulated in small quantities in organs (Figure 2(b)): the sum of all radioactivity measured in the bladder, brain, heart, kidneys, liver, lungs, spleen, and thyroid gland was 11 %ID, suggesting that free 125 I may be quickly excreted. Indeed, free 125 I was predominantly found in the bladder, kidneys and liver (2, 1, and 2 %ID; Figure 2(b)), and the thyroid (4 %ID; To estimate the free 125 I "concentration" in each site, we calculated the %ID per gram of tissue (%ID/g; Figure 2(c)). The highest concentrations were found in the thyroid and bladder (39 and 21 % ID/g; Figure 2(c)), as expected. The liver concentration was only 2 %ID/g, indicating that accumulation in this organ is mostly due to liver's large size and open vasculature rather than specificity. The brain, heart, kidneys, lungs, and spleen also had low free 125 I amounts and concentrations ( Figure 2(b,c)).
Then, we aimed to verify that this label can be detected as free 125 I in tissue samples. The organs from the described experiment were homogenized and mixed with TCA, while blood samples were directly mixed with this reagent. After precipitation and centrifugation, an aliquot of the supernatant was assessed for free 125 I. This demonstrated that all 125 I present in the body was detected as a free species: 100% and ≥84% of all 125 I in blood (Figure 2(d)) and organs ( Figure 2(e)), respectively. This was independent of the absolute level or concentration of 125 I found: 100% of all 125 I detected in blood was in free form although at early and late time points the biodistribution of the total radiolabel was quite different (46 %ID at 1 min vs. 13 %ID at 60 min); similarly, organs with higher or lower 125 I concentrations (39 %ID/g in the thyroid vs. 0.6% ID/g in the brain) contained 90% free 125 I. Thus, the method employed enables accurate detection of any free radiolabel present and may be used to correct total CPM data for the free radiolabel fraction found in blood and organs.
2.3 | Implementation of control, free 125 I corrections on the biodistribution of 125 I-Ab/3DNA Free 125 I biodistribution ranged broadly among different organs, that is, from 0.6 %ID/g in the brain to 39 %ID/g in the thyroid (Figure 2(c)).
This indicates that the presence of free label in a DDS formulation needs to be corrected by its own free 125 I biodistribution and, as such, the %ID observed in each organ ( Figure 2) could be used to correct carrier biodistribution data. Using the thyroid example, TCA implementation on the formulation to be injected would quantify the preadministration free 125 I label, 4% of which should distribute to the thyroid per Figure 2(b). The free 125 I CPM in this gland must be subtracted from the total CPM empirically measured for this tissue to determine the carriers found in the gland without any free 125 I label.
To test this, we injected mice with 125 I-Ab/3DNA as previously, 35,37,38 yet co-administered with a known amount (40-50%) of free 125 I label. We used a nonspecific Ab-oligo to avoid biodistribution changes and cellular uptake driven by specific targeting, which may complicate interpretation. Validating this Ab/3DNA model, our publication 37 shows that coupling of 3DNA to a specific Ab-oligo recognizing intercellular adhesion molecule 1 (ICAM-1), a protein preferentially expressed on the pulmonary endothelium, markedly increased lungspecific targeting compared to controls. 37 The circulation of the 125 I-Ab/3DNA + 125 I formulation (herein called 125 I-Ab/3DNA for simplicity) was assessed at various times after administration and organ bio-  (Table S1). Yet, contrary to free 125 I alone, 125 I-Ab/3DNA did not accumulate preferentially in the thyroid (2 %ID and 13 %ID/g; Figure 3(b,c) white bars) but in the liver (28 %ID and 25 %ID/g; Figure 3(b,c) white bars), as expected for a nanocarrier with an Ab coat. 3,37 These results describe the noncorrected data, but then, we implemented the explained correction ( Figure 3, black symbols and bars). Corrected data showed some changes for the circulation of 125 I-Ab/3DNA (44% increase at 60 min; 1.4-fold increase in AUC, Table S1) but, mainly, it lowered accumulation in the thyroid (by 94% for both the %ID and %ID/g of organ) and increased liver and spleen accumulation (by 92% and 82%, looking at the %ID parameter), expected changes for a nanosized formulation versus free 125 I.
In vitro generation of free 125 I from 125 I-Ab/3DNA in pre-and post-administration conditions. 125 I-Ab/3DNA was incubated with either control buffer (3% BSA in PBS) or tissue samples (blood and homogenized kidney or liver) from C57BL/6 mice. Percentage of free 125 I was calculated from the CPM corresponding to the total 125 I versus free 125 I in each sample as determined from radioactivity measurements in γ-counter prior and after trichloroacetic acid (TCA) precipitation, respectively. Data are mean ± S.D. (n = 3-4). *Compares 60 min versus 0 min, for each condition; $ compares blood, kidneys, and liver versus PBS/BSA, # compares kidneys and liver versus blood condition; (p < 0.05 by Student's t-test) To validate this correction, we performed TCA precipitation on the samples, to obtain the empirical level of free 125 I in each of them.
The level of free 125 I measured in blood was ≈15 %ID at 60 min ( Figure 4(a)), similar to that of free 125 I injected alone (Figure 2(a)). The t 1/2 , AUC, and MRT were 1.5 min, 203.6 ID%×min, and 2.2 min (Table S1), also similar to the free 125 I injection. The biodistribution varied broadly among organs (0.1 %ID in the heart and 3 %ID in the thyroid gland; Figure 4(b)), similar to free 125 I variability (Figure 2(b)).
However, unexpectedly, the biodistribution of free 125 I within the 125 I-Ab/3DNA injection differed from that of free 125 I control: %ID/g in kidneys decreased by 10%, both %ID and %ID/g in the lung decreased by 32% and 31%, %ID/g was reduced by 18% in the bladder and 42% in the thyroid (Figure 4(b,c)). Conversely, the %ID/g increased 17% for the spleen and both the %ID and %ID/g increased F I G U R E 3 Control free 125 I corrections of the 125 I-Ab/3DNA biodistribution. 125 I-Ab/3DNA was i.v. injected in C57BL/6 mice along with a known amount of free 125 I. Blood and organs were collected and weighed at the indicated times and measured in a γ-counter. Results were calculated from the total CPM measured in blood and organs and referred to as the total CPM of the injected dose (including 125 I-Ab/3DNA + free 125 I for noncorrected data), or they were corrected to subtract in each tissue the expected biodistribution of free 125 I obtained in Figure 2  by 386% and 335% for the liver. Therefore, this correction that assumes a similar biodistribution of free 125 I within the 125 I-Ab/3DNA as for the free 125 I injected alone is not accurate.
Additional controls were performed to demonstrate if free 125 I would interact with hemoglobin or albumin, as reported. 39,40 We incubated 125 I-Ab-oligo (the radiolabeled counterpart in 125 I-Ab/3DNA) containing 10% free 125 I with heparinized mouse blood or plasma. After 30 min, we performed TCA precipitation and found 11.8 ± 0.7% and 12.2 ± 0.75% free 125 I in these mixtures. Independently, free 125 I alone (100%) was incubated with whole blood or plasma, after which TCA precipitation rendered detection of 102.7 ± 2.0% and 110.8 ± 0.5% free 125 I. Mixing of nonlabeled (cold) Ab/3DNA with free 125 I followed by TCA precipitation, rendered 105.1 ± 0.4% free 125 I detection, demonstrating that all the free label is detected despite the presence of the carrier. Mixing of cold Ab/3DNA with 125 I-Ab-oligo containing 10% free 125 I lead to detection of 11.0 ± 0.4% free iodine, demonstrating that even if some 125 I-Ab-oligo counterpart were to detach from the 3DNA, this would not interfere with the ability to detect free 125 I.

| Implementation of empirical free 125 I corrections on the biodistribution of 125 I-Ab/3DNA
Therefore, free 125 I in blood and organ samples must be empirically measured within each experiment, to subtract this value from the total CPM in said samples. To prove this, out of the total CPM found in each blood and organ sample, we calculated which fraction corresponded to free 125 I, not true 125 I-Ab/3DNA ( Figure 5). Results show that much of the label found in blood was free 125 I (e.g., 29% of all CPM in this organ, at 60 min; Figure 5(a)), while in tissues this fraction varied broadly from 13% of all CPM in the spleen to 54% all CPM in the brain or 95% of all CPM in the bladder (Figure 5(b)).
The contribution of free 125 I to the total CPM of each tissue sample was highly variable, validating that the best possible correction for "clean" carrier biodistribution data is to subtract free 125 I counts in each organ from the total counts in said organ. Implementing this correction showed that the blood clearance profile remained similar, although a bit enhanced for the 60 min point ( Figure 5(c)). The t 1/2 , and MRT were 39.6 min and 57.1 min (Table S1). Organ accumulation greatly changed after free 125 I correction: accumulation in the bladder and the thyroid decreased by 3-fold, while liver and spleen accumulation increased by 1.5-fold ( Figure 5(d,e)).
Then, we injected mice with 125 I-Ab/3DNA mixed with a low amount of free 125 I (10% vs. 40-50% in the prior experiment). The contribution of free 125 I CPM to the total CPM found in each tissue was relatively low (Figure 6(a)) and very reduced for the bladder and thyroid which accumulate the free label compared to the high contribution of free 125 I CPM of the free 125 I control injection (Figure 2(e)). The biodistribution of 125 I-Ab/3DNA containing low free 125 I level was determined and compared to 125 I-Ab/3DNA with high 125 I level. For both, corrected and noncorrected data ratio (fold difference) was compared.
There was little difference between the corrected and noncorrected biodistribution of the formulation carrying low free 125 I (yffi1 in Figure 6 (b); black bars), unlike the formulation with high level of this radiolabel (y ≠ 1 in Figure 6(b); white bars). Therefore, this method is valid to obtain accurate biodistribution data in the presence of free radiolabel which may arise from metabolism or degradation in the body.

| Method implementation using targeted versus nonspecific 125 I-Ab/3DNA and comparing different biodistribution times
We next used this method to determine the biodistribution of targeted 125 I-Ab/3DNA, using an Ab-oligo recognizing ICAM-1 ( Figure 7). This is a cell-surface protein preferentially expressed on the lung endothelium, which provides lung-specific targeting. 37 This formulation had 179 ± 6 nm diameter, 0.25 ± 0.017 PDI, and −38 ± 0.6 mV ζ-potential, similar to IgG/3DNA described above. Both for noncorrected and corrected data, 125 I-anti-ICAM/3DNA disappeared faster from the circulation than the nonspecific formulation ( Figure 7(a)). For example, for corrected data, t 1/2 and MRT were F I G U R E 5 Contribution of free 125 I to the total 125 I signal found in blood and organs after 125 I-Ab/3DNA injection and respective biodistribution correction. C57BL/6 mice were injected with 125 I-Ab/3DNA containing a known amount of CPM of free 125 I. (a) Blood samples were collected at the indicated times post injection and (b) organs were collected at sacrifice at 60 min for CPM measurements in γ-counter. In both cases, total CPM and free 125 I CPM were measured prior to and after TCA precipitation, so that the fraction of free 125 I over the total CPM in each body compartment was calculated. (c) Circulation and (d) biodistribution of 125 I-Ab/3DNA expressed as percentage of the injected dose (% ID), which were calculated from the total CPM measured and referred to the total CPM of the injected dose (noncorrected), or corrected to subtract free 125 I CPM in each tissue as well as the dose. (e) Similar data but referred as %ID per gram organ (%ID/g) to show organ concentration. Data are mean ± S.D. (n ≥ 5 mice). *Compares corrected to noncorrected data (p < 0.05 by Student's t-test) F I G U R E 6 Free 125 I content and corrected to noncorrected ratio of the biodistribution of 125 I-Ab/3DNA containing minimal free 125 I. (a) 125 I-Ab/3DNA containing minimal (10%) free 125 I was i.v. injected in C57BL/6 mice. Total CPM and free 125 I CPM were measured prior to and after TCA precipitation, so that the fraction of free 125 I over the total CPM in each body compartment was calculated. (b) 125 I-Ab/3DNA with 10% versus 45% free 125 I were i.v. injected, and organs were collected and weighed at the indicated times and measured in a γ-counter prior to and after TCA precipitation to calculate the biodistribution as the percentage of the injected dose (%ID). This was done from noncorrected data (total CPM in tissue/total CPM in the injected dose) or corrected values ([totalfree 125 I CPM in tissue]/[totalfree 125 I CPM in the injected dose]), from where the corrected to non-corrected ratio was calculated. Data are mean ± S.D. (n ≥ 5 mice) 0.36 min and 0.57 min compared to 39.6 min and 57.1 min for 125 I-IgG/3DNA (Table S1). This is known to be due to fast targeting of lung ICAM-1, confirmed here. For example, corrected data showed 122 %ID/g of lung for the targeted formulation versus 4 %ID/g of lung for the control (Figure 7(b)). Biodistribution in the liver and bladder, examples of metabolic clearance and excretion organs, was much lower (33 and 0.5 %ID/g for anti-ICAM/3DNA) and closer to IgG/3DNA levels (24 and 0.1 %ID/g for IgG/3DNA). A more specific lung targeting for anti-ICAM/3DNA over IgG/3DNA was found by using this method: 22-fold difference using noncorrected data versus 33-fold difference using corrected data. High free 125 I found in the lung for IgG/3DNA versus low free 125 I for anti-ICAM/3DNA (27 vs. 4% of all lung signal) explains this difference (Figure 7(c)).
Free 125 I found in the liver and bladder was similar for both formulations (e.g., 7.8 and 7.6% of all liver signal), the latter organ carrying much higher levels 85 and 75% of all bladder signal), as expected.
Finally, using IgG/3DNA, we implemented this method to determine biodistribution changes that may result from time-dependent degradation in the body (Figure 8). Noncorrected data (Figure 8(a)) showed no change in the kidneys between 30 min and 60 min after injection (3.8 and 3.3 %ID/g), increased bladder levels (1 and 9 %ID/g), and similar liver levels (26 and 25 %ID/g). This is counterintuitive since nanoformulations are large enough to avoid kidney filtration and not expected to be excreted into the bladder, while the liver is expected to collect circulating DDSs over time. This was observed after data correction: from 24 to 44 %ID/g increase in the liver and similar levels (0.1 and 1.5 %ID/g) in the bladder (Figure 8(b)). Also  (Figure 8(c)), free 125 I increased in the liver (8 to 19%) with time, as expected due to hepatic metabolization.

| DISCUSSION
Radiotracing using γ emitters is considered the gold standard for biodistribution measurements as it provides high accuracy, sensitivity, and throughput abilities. 6,7 However, as for other labeling methods, 6 biodistribution data can be confounded by potential tracing of free-label generated prior to or after administration of biodegradable DDS. Hence, biodistribution studies could benefit from means to determine and correct for these artifacts. In this study, we employed TCA precipitation on blood and tissue samples to quantify the present or generated free radiolabel present or generated and correct biodistribution data accordingly.
We showed that some free 125 I is released from 125 I-Ab/3DNA upon incubation in vitro with buffer, blood or tissues (Figure 1), validating the need for correction methods. Next, using in vivo injection of free 125 I, we showed each organ accumulated different radiolabel levels ( Figure 2). The thyroid accumulated the highest 125 I concentration, likely due to the sodium-iodide symporter (NIS). 41,42 Although kidneys also express NIS, 42 we did not see high accumulation here, possibly due to 125 I secretion in the urine. In fact, the bladder accumulated the second greatest free 125 I concentration (Figure 2(c)). Other organs showed low free 125 I concentrations (Figure 2(c)), although the absolute accumulation was more prominent in the liver (Figure 2(b)), perhaps due to its fenestrated endothelium and large volume. Hence, the level of free 125 I widely varies among the different body compartments, justifying the need to correct 125 I data for free label in each compartment. The implemented method allowed precise classification of all detected 125 I as a free species in the organs tested (Figure 2(d,e)), indicating that free 125 I was not incorporated in relevant amounts into body molecules, which would otherwise be precipitated by TCA.
Then, we tested the method using 125 I-Ab/3DNA containing a known amount of free 125 I, which demonstrated the confounding effects of free radiolabel on biodistribution results. We used the biodistribution of free 125 I from the control experiment to correct for the presence of the free 125 I in the blood and organs of these animals ( Figure 3). This showed an increased accumulation in the liver and spleen and a thyroid decrease, expected for a nanodevice. Unexpectedly, we observed the biodistribution of free 125 I co-injected with 125 I-Ab/3DNA differed from the biodistribution of free 125 I administered alone (Figure 4). Speculatively, 125 I-Ab/3DNA may compete with free 125 I for access to certain organs or free 125 I may arise from 125 I-Ab/3DNA degradation. Since the liver plays a role in the degradation, increased free 125 I in this organ in animals injected with 125 I-Ab/ 3DNA + free 125 I may be due to some 125 I-Ab/3DNA degradation in the liver, as suggested by ex vivo 125 I release from 125 I-Ab/3DNA in liver homogenates (Figure 1). This phenomenon would be less significant in organs unrelated to clearance/degradation, such as the heart.
Additionally, as other DDSs, 43 125 I-Ab/3DNA may form a protein corona containing albumin, which is known to carry 125 I. 39 If an albumin corona forms on 125 I-Ab/3DNA, some free 125 I may associate to it in the body. Since 125 I-Ab/3DNA accumulates significantly in the liver, it may carry here free 125 I via protein corona, accounting for the different biodistribution of free 125 I co-injected with 125 I-Ab/3DNA. This phenomenon indicates the biodistribution of 125 I-Ab/3DNA cannot be corrected using the biodistribution of free 125 I alone, but free 125 I must be measured in each organ sample within the same experiment. The comparison of corrected vs. noncorrected biodistribution of 125 I-Ab/3DNA showed clear differences in organ biodistribution ( Figure 5(d)), there was lower accumulation in organs which predominantly take free radiolabel but are not expected to accumulate 125 I-Ab/3DNA (bladder and thyroid; Figure 5(d,e)), removing this confounding signal. Accumulation of 125 I-Ab/3DNA in the liver and spleen increased upon correction (Figure 5(d)), in agreement with reports on Ab/nanovehicles of similar size and ζ-potential (30-50% ID/g). 19 In organs where free 125 I did not substantially accumulate (brain, heart, lungs; Figures 2(b,c) and 4(b,c)), correction did not impact the biodistribution data ( Figure 5(d,e)). This correction significantly impacted the comparative biodistribution of targeted versus a nontargeted formulations, anti-ICAM/3DNA versus IgG/3DNA in this case (Figure 7(b)), for example, from 22-to 33-fold difference, highlighting the relevance of this method. The method also demonstrated value in better determining changes in biodistribution over time (Figure 8(a,b)) and in vivo degradation patterns (Figure 8(c)). This sensitivity is facilitated by the easiness of the implemented method, for example, samples extracted from the animal can be processed at   4.4 | Label detachment from 125 I-Ab/3DNA in buffer, blood, and organ homogenates C57BL/6 mice were anesthetized to collect 500 μl blood samples from the retro-orbital sinus and sacrificed to extract the kidneys and liver, which were homogenized at 28,000 rpm using a Kinematica Polytron™ PT 3100D homogenizer (Kinematica, Lucerne, Switzerland). 125 I-Ab/3DNA was added to the blood or organ homogenates, or control PBS-3% BSA, and incubated at 37 C. To detect free 125 I, samples were mixed with 3% BSA in PBS up to 1 ml, then precipitated by adding 200 μl TCA (17% v/v final concentration), incubation for 15 min at 4 C, and centrifugation at 2687g for 10 min. Using a γ-counter, free 125 I CPM was determined from 600 μl of the sample supernatants and total CPM of the samples, measured prior to TCA precipitation, was used to calculate the percent of free 125 I released compared to time = 0 from 125 I-Ab/3DNA as follows:

| Statistical Analysis
Data were calculated as mean ± standard deviation (S.D.). Experiments encompassed n ≥ 4 mice. Significance was determined using the Student's unpaired t-test, assuming a p-value of 0.05.

| CONCLUSION
Results indicate the presence of free 125 I radiolabel in biodistribution samples, which could be generated due to DDS degradation prior to or after administration, needs to be accounted within the same experiment for each organ or blood sample to obtain accurate biodistribution results. This can be achieved by implementing TCA precipitation of said samples, permitting the accurate classification of signals originating from free radiolabel vs. radiolabeled carrier. This method is valuable to characterize DDS parameters such as the presence of free radiolabel in the dose to be administered, interaction of free radiolabel with DDS itself, and in vivo stability or degradation profiles of the formulation, for example, in different organs that may have different capacities to metabolize the labeled DDS components.